Modified alpha hemolysin polypeptides and methods of use

ABSTRACT

Provided herein are alpha hemolysin polypeptides comprising modified amino acid sequences that can reduce the rate of translocation of a polymer. Also provided herein are apparatuses and devices comprising modified hemolysin polypeptides. Also provided herein are methods of using modified alpha hemolysin proteins for use in characterizing and/or sequencing a polymer or for use as molecular sensors.

RELATED PATENT APPLICATIONS

This patent application is a 35 U.S.C. 371 national phase patentapplication of PCT/US2013/076698, filed on Dec. 19, 2013, entitledMODIFIED ALPHA HEMOLYSIN POLYPEPTIDES AND METHODS OF USE, namingGeoffrey A. Barrall, Eric N. Ervin and Prithwish Pal as inventors, whichclaims the benefit of U.S. provisional patent application No. 61/740,322filed on Dec. 20, 2012, entitled MODIFIED ALPHA HEMOLYSIN PROTEIN PORESAND METHODS OF USE, naming Geoffrey A. Barrall, Eric N. Ervin andPrithwish Pal as inventors. The entire content of the foregoingapplication is incorporated herein by reference, including all text,tables and drawings.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.1R01HG005095 and 2R44HG004466 awarded by the National Institutes ofHealth, specifically the National Human Genome Research institute. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 29, 2014, isnamed EBS-1007-PC_SL.txt and is 9,124 bytes in size.

FIELD

The technology herein relates, in part, to modified alpha hemolysinpolypeptides that transport polymers across a membrane.

BACKGROUND

Hemolysins are members of a family of protein toxins that are producedby a wide variety of organisms. Some hemolysins, for example alphahemolysins, can disrupt the integrity of a cell membrane (e.g., a hostcell membrane) by forming a pore or channel in the membrane. Pores orchannels that are formed in a membrane by pore forming proteins can beused to transport certain polymers (e.g., polypeptides orpolynucleotides) from one side of a membrane to the other.

SUMMARY

Provided herein, in certain aspects, is a polypeptide comprising amodified alpha hemolysin amino acid sequence comprising one or moreamino acid substitutions at one or more positions corresponding topositions 1-109 and 149-293 of the amino acid sequence of SEQ ID NO: 1,where each of the one or more amino acid substitutions independently isto (i) a non-native hydrophobic amino acid, or (ii) a non-nativearomatic amino acid, or (iii) a non-native aromatic and hydrophobicamino acid. A modified alpha hemolysin polypeptide, the latter of whichis also referred to as a modified alpha hemolysin protein pore herein,generally comprises, consists essentially of or consists of a modifiedalpha hemolysin amino acid sequence. An amino acid sequence of awild-type (i.e., native, unmodified) alpha hemolysin polypeptide fromStaphylococcus aureus is provided herein as SEQ ID NO: 1. In certainaspects a modified alpha hemolysin amino acid sequence comprises anamino acid modification at one or more positions corresponding topositions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 97, 99,101, 103, 105, 107, 109, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 225, 227, 229, 231 or 233 of SEQ ID NO: 1. In certain aspectsthe one or more positions correspond to positions 105, 107, 109, 149,151 or 153 of SEQ ID NO: 1. In certain aspects the one or more positionscorrespond to positions 107, 109, 149 or 151 of SEQ ID NO: 1. In certainaspects the one or more positions correspond to positions 109 or 149 ofSEQ ID NO: 1.

In certain aspects the modified alpha hemolysin amino acid sequence alsocomprises one or more substitutions located within a beta barrel of thealpha hemolysin polypeptide, where the one or more positions for the oneor more substitutions in the beta barrel sometimes are chosen from oneor more positions corresponding to positions 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 and 147of SEQ ID NO: 1. In certain aspects the modified alpha hemolysin aminoacid sequence comprises at least two, at least three, at least four orat least five amino acid substitutions located in the beta barrel.

In certain aspects, a modified alpha hemolysin polypeptide is configuredto translocate a polymer at least 20% slower than a reference alphahemolysin polypeptide or at least 20% slower than a reference alphahemolysin protein comprising one or more amino acid substitutionslocated in a beta barrel.

Also provided herein is a method of sequencing a polymer with a modifiedalpha hemolysin polypeptide comprising (a) contacting a polymer with amodified alpha hemolysin polypeptide, wherein the modified alphahemolysin polypeptide comprises an amino acid sequence of a referencealpha hemolysin polypeptide comprising one or more amino acidsubstitutions, and (b) determining the sequence of the polymer accordingto one or more electrical changes across or through the modified alphahemolysin polypeptide. A modified alpha hemolysin polypeptide describedherein can be utilized in this method.

Also provided herein is a method of increasing a translocation time of apolymer through an alpha hemolysin polypeptide, comprising (a)contacting a polymer with a modified alpha hemolysin polypeptide,wherein the modified alpha hemolysin polypeptide comprises an amino acidsequence of a reference alpha hemolysin polypeptide comprising one ormore amino acid substitutions, and (b) determining a first translocationtime of the polymer through the modified alpha hemolysin polypeptide,wherein the first translocation time of the polymer through the modifiedalpha hemolysin polypeptide is at least 20% longer than a secondtranslocation time of the polymer through the reference alpha hemolysinpolypeptide. A modified alpha hemolysin polypeptide described herein canbe utilized in this method.

Also provided herein is a method of translocating a polymer through amodified alpha hemolysin polypeptide, comprising (a) substituting one ormore amino acids of a reference alpha hemolysin polypeptide, where amodified alpha hemolysin polypeptide is generated, and (b) contactingthe modified alpha hemolysin polypeptide with a polymer, wherein thepolymer translocates through the modified alpha hemolysin polypeptide. Amodified alpha hemolysin polypeptide described herein can be utilized inthis method.

Also provided herein in certain aspects is a nanopore device comprisinga modified alpha hemolysin polypeptide, such as a modified alphahemolysin polypeptide described herein.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain aspects and embodiments of thetechnology and are not limiting. For clarity and ease of illustration,the drawings are not made to scale and, in some instances, variousaspects may be shown exaggerated or enlarged to facilitate anunderstanding of particular embodiments and aspects.

FIGS. 1-13 below each show three histograms for the data taken with amodified alpha hemolysin polypeptide (i.e., modified alpha hemolysinprotein pore (e.g., Figures D-F) and the corresponding unmodified alphahemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid (e.g., Figures A-C).

FIGS. 1A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide 4SSDKMS (e.g., without the at least one native amino acid substituted withthe non-native amino acid)(FIGS. 1A-1C; 2D density translocationstatistics: Tmax=102 μsec and open channel=−238/+254 pA) and a modifiedalpha hemolysin polypeptide YY-4S SDKMS (FIGS. 1D-1F; 2D densitytranslocation statistics: Tmax C100 (SEQ ID NO: 4)−3′=362 μsec and openchannel=−232/+251 pA). FIGS. 1A and 1D show amplitude histograms, FIGS.1B and 1E show duration histograms and FIGS. 1C and 1F show standarddeviation histograms for the indicated alpha hemolysin proteins.

FIGS. 2A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide 4SSDKMS (e.g., without the at least one native amino acid substituted withthe non-native amino acid)(FIGS. 2A-2C; 2D density translocationstatistics: Tmax=102 μsec and open channel=−238/+254 pA) and a modifiedalpha hemolysin polypeptide 4Y-4S SDKMS (FIGS. 2D-2F; 2D densitytranslocation statistics: Tmax=398 μsec and open channel=−231/+241 pA).FIGS. 2A and 2D show amplitude histograms, FIGS. 2B and 2E show durationhistograms and FIGS. 2C and 2F show standard deviation histograms forthe indicated alpha hemolysin proteins.

FIGS. 3A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide4S+SDKMS (e.g., without the at least one native amino acid substitutedwith the non-native amino acid)(FIGS. 3A-3C; 2D density translocationstatistics: Tmax=102 μsec and open channel=−238/+254 pA) and a modifiedalpha hemolysin polypeptide V149W-4S SDKMS (FIGS. 3D-3F; 2D densitytranslocation statistics: Tmax=271 μsec and open channel=−231/+254 pA).FIGS. 3A and 3D show amplitude histograms, FIGS. 3B and 3E show durationhistograms and FIGS. 3C and 3F show standard deviation histograms forthe indicated alpha hemolysin proteins.

FIGS. 4A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide 4SSDKMS (e.g., without the at least one native amino acid substituted withthe non-native amino acid)(FIGS. 4A-4C; 2D density translocationstatistics: Tmax=102 μsec and open channel=−238/+254 pA) and a modifiedalpha hemolysin polypeptide YW-4S SDKMS (FIGS. 4D-4F; 2D densitytranslocation statistics: Tmax=1135 μsec and open channel=−234/+250 pA).FIGS. 4A and 4D show amplitude histograms, FIGS. 4B and 4E show durationhistograms and FIGS. 4C and 4F show standard deviation histograms forthe indicated alpha hemolysin proteins.

FIGS. 5A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide4S+SDKMS (e.g., without the at least one native amino acid substitutedwith the non-native amino acid)(FIGS. 5A-5C; 2D density translocationstatistics: Tmax=102 μsec and open channel=−238/+254 pA) and a modifiedalpha hemolysin polypeptide WY-4S SDKMS (FIGS. 5D-5F; 2D densitytranslocation statistics: Tmax=919 μsec and open channel=−235/+251 pA).FIGS. 5A and 5D show amplitude histograms, FIGS. 5B and 5E show durationhistograms and FIGS. 5C and 5F show standard deviation histograms forthe indicated alpha hemolysin proteins.

FIGS. 6A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide4S+SDKMS (e.g., without the at least one native amino acid substitutedwith the non-native amino acid)(FIGS. 6A-6C; 2D density translocationstatistics: Tmax=102 μsec and open channel=−238/+254 pA) and a modifiedalpha hemolysin polypeptide 107W T109W-4S SDKMS (FIGS. 6D-6F; 2D densitytranslocation statistics: Tmax=249 μsec and open channel=−243/+259 pA).FIGS. 6A and 6D show amplitude histograms, FIGS. 6B and 6E show durationhistograms and FIGS. 6C and 6F show standard deviation histograms forthe indicated alpha hemolysin proteins.

FIGS. 7A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide 4SL135I D127K (e.g., without the at least one native amino acidsubstituted with the non-native amino acid)(FIGS. 7A-7C; 2D densitytranslocation statistics: Tmax C100 (SEQ ID NO: 4)=100 μsec and openchannel=−232/+272 pA) and a modified alpha hemolysin polypeptide YY-4SL135I D127K (FIGS. 7D-7F; 2D density translocation statistics: Tmax C100(SEQ ID NO: 4)−3′=420 μsec and open channel=−232/+272 pA). FIGS. 7A and7D show amplitude histograms, FIGS. 7B and 7E show duration histogramsand FIGS. 7C and 7F show standard deviation histograms for the indicatedalpha hemolysin proteins.

FIGS. 8A-F show histograms of translocations statistics for polyA100(SEQ ID NO: 5) translocating through alpha hemolysin polypeptide 4SL135I T125Q D127K (e.g., without the at least one native amino acidsubstituted with the non-native amino acid)(FIGS. 8A-8C; 2D densitytranslocation statistics: Tmax=278 μsec and open channel=−242/+275 pA)and a modified alpha hemolysin polypeptide YY-4S L135I T125Q D127K(FIGS. 8D-8F; 2D density translocation statistics: Tmax=589 μsec andopen channel=−230/+269 pA). FIGS. 8A and 8D show amplitude histograms,FIGS. 8B and 8E show duration histograms and FIGS. 8C and 8F showstandard deviation histograms for the indicated alpha hemolysinproteins.

FIGS. 9A-F show histograms of translocations statistics for polyA100(SEQ ID NO: 5) translocating through alpha hemolysin polypeptide 4SL135I T125S D127K (e.g., without the at least one native amino acidsubstituted with the non-native amino acid)(FIGS. 9A-9C; 2D densitytranslocation statistics: Tmax=156 μsec and open channel=−234/+276 pA)and a modified alpha hemolysin polypeptide YY-4S L135I T125S D127K(FIGS. 9D-9F; 2D density translocation statistics: Tmax=1170 μsec andopen channel=−232/+278 pA). FIGS. 9A and 9D show amplitude histograms,FIGS. 9B and 9E show duration histograms and FIGS. 9C and 9F showstandard deviation histograms for the indicated alpha hemolysinproteins.

FIGS. 10A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide 4SL135I T125S D127K (e.g., without the at least one native amino acidsubstituted with the non-native amino acid)(FIGS. 10A-10C; 2D densitytranslocation statistics: Tmax=58 μsec and open channel=−235/+281 pA)and a modified alpha hemolysin polypeptide YY-4S L135I T125S D127K(FIGS. 10D-10F; 2D density translocation statistics: Tmax=275 μsec andopen channel=−241/+279 pA). FIGS. 10A and 10D show amplitude histograms,FIGS. 10B and 10E show duration histograms and FIGS. 10C and 10F showstandard deviation histograms for the indicated alpha hemolysinproteins.

FIGS. 11A-F show histograms of translocations statistics for polyA100(SEQ ID NO: 5) translocating through alpha hemolysin polypeptide 4S N3TL135I T125N D127K (e.g., without the at least one native amino acidsubstituted with the non-native amino acid)(FIGS. 11A-11C; 2D densitytranslocation statistics: Tmax=163 μsec and open channel=−232/+278 pA)and a modified alpha hemolysin polypeptide YY-4S N3T L135I T125N D127K(FIGS. 11D-11F; 2D density translocation statistics: Tmax=686 μsec andopen channel=−240/+278 pA). FIGS. 11A and 11D show amplitude histograms,FIGS. 11B and 11E show duration histograms and FIGS. 11C and 11F showstandard deviation histograms for the indicated alpha hemolysinproteins.

FIGS. 12A-F show histograms of translocations statistics for polyA100(SEQ ID NO: 5) translocating through alpha hemolysin polypeptide E111DM113S N2S L135I D127K (e.g., without the at least one native amino acidsubstituted with the non-native amino acid)(FIGS. 12A-12C; 2D densitytranslocation statistics: Tmax=169 μsec and open channel=−230/+247 pA)and a modified alpha hemolysin polypeptide YY-E111D M113S N2S L135ID127K (FIGS. 12D-12F; 2D density translocation statistics: Tmax=400 μsecand open channel=−217/+238 pA). FIGS. 12A and 12D show amplitudehistograms, FIGS. 12B and 12E show duration histograms and FIGS. 12C and12F show standard deviation histograms for the indicated alpha hemolysinproteins.

FIGS. 13A-F show histograms of translocations statistics for polyC100(SEQ ID NO: 4) translocating through alpha hemolysin polypeptide E111DM113S N2S L135I D127K (e.g., without the at least one native amino acidsubstituted with the non-native amino acid)(FIGS. 13A-13C; 2D densitytranslocation statistics: Tmax polyC100 (SEQ ID NO: 4)−3′=79 μsec andopen channel=−240/+255 pA) and a modified alpha hemolysin polypeptideYY-E111D M113S N2S L135I D127K (FIGS. 13D-13F; 2D density translocationstatistics: Tmax polyC100 (SEQ ID NO: 4)−3′=314 μsec and openchannel=−218/+242 pA). FIGS. 13A and 13D show amplitude histograms,FIGS. 13B and 13E show duration histograms and FIGS. 13C and 13F showstandard deviation histograms for the indicated alpha hemolysinproteins.

DETAILED DESCRIPTION

Hemolysins and Alpha Hemolysin

Hemolysins are exotoxins produced by bacteria that cause lysis of redblood cells in vitro or in vivo. Visualization of hemolysis of red bloodcells in agar plates facilitates the categorization of some pathogenicbacteria such as Streptococcus and Staphylococcus. Although the lyticactivity of some hemolysins on red blood cells may be important fornutrient acquisition or for causing certain conditions such as anemia,many hemolysin-producing pathogens do not cause significant lysis of redblood cells during infection. Although hemolysins are able to lyse redblood cells in vivo, the ability of hemolysins to target other cells,including white blood cells, often accounts for the effects ofhemolysins in the host. Many hemolysins are pore forming proteins.

A non-limiting example of a hemolysin porin protein useful for insertioninto lipid bilayers is alpha hemolysin, sometimes also referred to asalpha toxin. Alpha hemolysin forms a heptameric beta-barrel inbiological membranes. Alpha hemolysin is secreted as a monomer thatbinds to the outer membrane of susceptible cells. Upon binding, themonomers oligomerize to form a water-filled transmembrane channel thatfacilitates uncontrolled permeation of water, ions, and small organicmolecules. Rapid discharge of vital molecules, such as ATP, dissipationof the membrane potential and ionic gradients, and irreversible osmoticswelling leading to rupture or lysis of the cell wall, frequentlycausing death of the host cell. This pore-forming property has beenidentified as a major mechanism by which protein toxins cause damage tocells.

However, the ability to use wild-type hemolysin polypeptides foranalysis of polymers (e.g., for polymer sequencing) presents certaintechnological challenges. For example, certain polymers translocate tooquickly through a wild-type alpha hemolysin pore to be accuratelyanalyzed or sequenced. Presented herein are modified alpha hemolysinpolypeptides that demonstrate reduced rates of polymer translocationthat can, for example, provide for more efficient and accurate analysisof translocating polymers.

An amino acid sequence of any suitable pore forming hemolysin protein,homologous protein, or pore forming portion thereof can be modified bymethods described herein to generate a modified alpha hemolysin aminoacid sequence. Non-limiting examples of pore forming hemolysins includelisteriolysin O (e.g., from Listeria monocytogenes), alpha toxin oralpha hemolysin (e.g., from Staphylococcus aureus), PVL cytotoxin (e.g.,from Staphylococcus aureus), cytolysin A (e.g., from E. coli),substantially homologous proteins thereof, pore forming portionsthereof, the like and combinations thereof (e.g., chimeric variantsthereof and/or heteromers thereof).

Polypeptides Comprising a Modified Alpha Hemolysin Amino Acid Sequence

In certain embodiments, a polypeptide comprises a modified alphahemolysin amino acid sequence. In some embodiments, a polypeptidecomprising a modified alpha hemolysin amino acid sequence is modifiedrelative to a reference alpha hemolysin polypeptide. In someembodiments, a polypeptide comprising a modified alpha hemolysin aminoacid sequence is modified relative to a wild type alpha hemolysinpolypeptide. In some embodiments a modified alpha hemolysin amino acidsequence comprises an amino acid sequence of a wild-type alpha hemolysinprotein or of a wild-type alpha hemolysin protein that has beenmodified. For example a modified alpha hemolysin protein may compriseone or more amino acid substitutions, deletions or insertions prior tobeing modified as described herein. A wild-type hemolysin protein can beany alpha hemolysin protein found in nature. In some embodiments awild-type alpha hemolysin protein comprises an amino acid sequence withat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 1. In some embodimentsa wild-type alpha hemolysin protein comprises an amino acid sequencewith at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequencehomology to the amino acid sequence of SEQ ID NO: 1. In some embodimentsa wild-type alpha hemolysin protein consists of an amino acid sequenceof SEQ ID NO: 1.

In some embodiments a “modified alpha hemolysin amino acid sequence”refers to a modification of an amino acid sequence of an alpha hemolysinprotein comprising one or more amino substitutions in a beta barrelregion. The terms “modified alpha hemolysin polypeptide,” “modifiedalpha hemolysin protein” and “modified alpha hemolysin protein pore”have the same meaning herein, are used interchangeably herein and referherein to a protein (e.g., a polypeptide) comprising a modified alphahemolysin amino acid sequence. A modified alpha hemolysin amino acidsequence can comprise any suitable modification of an amino acidsequence, non-limiting examples of which include one or more amino acidsubstitutions, amino acid modifications (e.g., substitution of an aminoacid with a modified or non-standard amino acid), deletions of one ormore amino acids, insertions of one or more amino acids, the like orcombinations thereof. Standard amino acids include Alanine, Cysteine,Aspartic acid, Glutamic acid, Phenylalanine, Glycine, Histidine,Isoleucine, Lysine, Leucine, Methionine, Asparagine, Proline, Glutamine,Arginine, Serine, Threonine, Valine, Tryptophan and Tyrosine which canbe represented herein by their standard IUPAC single letter or threeletter abbreviations. Non-limiting examples of non-standard amino acidsinclude α-Amino-n-butyric acid, Norvaline, Norleucine, Alloisoleucine,t-leucine, α-Amino-n-heptanoic acid, Pipecolic acid,α,β-diaminopropionicacid, α,γ-diaminobutyric acid, Ornithine, Allothreonine, Homocysteine,Homoserine, β-Alanine, β-Amino-n-butyric acid, β-Aminoisobutyric acid,γ-Aminobutyric acid, α-Aminoisobutyric acid, isovaline, Sarcosine,N-ethyl glycine, N-propyl glycine, N-isopropyl glycine, N-methylalanine, N-ethyl alanine, N-methyl β-alanine, N-ethyl β-alanine,isoserine, αhydroxy-γ-aminobutyric acid, Selenocysteine, Pyrrolysine andthe like.

Methods of modifying an amino acid sequence are well known and describedherein. Any suitable method can be used to modify an amino acid sequenceof a polypeptide. In some embodiments a nucleotide sequence is modified,which modified nucleic acid is subsequently used to express a modifiedprotein. Non-limiting examples of methods that can be used to modify anucleic acid sequence included PCR based site directed mutagenesistechniques (e.g., overlap extension PCR, round the horn, site directedmutagenesis, the like and combinations thereof), chemical DNA synthesis,and methods described in Maniatis, T., E. F. Fritsch and J. Sambrook(1982) Molecular Cloning: a Laboratory Manual; Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. Non-limiting examples of commercialkits that can be used to modify a nucleic acid sequence and/or aminoacid sequence include QuickChange kit (Stratagene, San Diego, Calif.,USA), Erase-a-base and pAlter-Max Vector (Promega, Sunnyvale, Calif.,USA), In-Fusion® HD Plus Complete Cloning System (Clontech Laboratories,Inc., A Takara Bio Company, Mountain View, Calif., USA), GeneMorph® IIEZClone Domain Mutagenesis Kit (Agilent Technologies, Santa Clara,Calif., USA), and Phusion Site-Directed Mutagenesis Kit (ThermoScientific, Waltham, Mass. USA). A modified nucleic acid can be used toexpress a modified polypeptide comprising a modified amino acid sequenceby any suitable method. Non-limiting examples of methods that can beused to generate a modified polypeptide comprising a modified amino acidsequence included Buculovirus expression systems, adenovirus expressionsystems, prokaryotic expression systems, phage expression systems,mammalian cell expression systems (e.g., 293 cell expression systems),yeast expression systems, in vitro translation systems, coupled in vitrotranscription/translation systems, the like and combinations thereof.Non-limiting examples of commercial protein expression systems that canbe used to express a modified polypeptide comprising a modifiedpolypeptide amino acid sequence include Gateway Nova, pET expressionsystems and Bac Magic expression systems (EMD Millipore, Temecula,Calif. USA), VariFlex expression systems (Agilent Technologies, SantaClara, Calif., USA) and Bac-to-Bac Baculovirus Expression System,ViraPower Adenoviral Gateway Expression Kit, Expressway Maxi Cell-FreeE. coli Expression System, and Retic Lysate IVT Kit (Life Technologies,Carlsbad, Calif. USA).

In some embodiments, a modified alpha hemolysin amino acid sequence ismodified in reference to the 293 amino acid sequence of an unmodifiedalpha hemolysin, provided herein as SEQ ID NO: 1. In some embodiments, amodified alpha hemolysin amino acid sequence is modified in reference tothe positions, or aligned positions, of the 293 amino acids of alphahemolysin of SEQ ID NO: 1. An amino acid in a modified amino acidsequence generally “corresponds to” an aligned amino acid when themodified amino acid sequence is aligned to the amino acid sequence ofSEQ ID NO: 1. For example, a modification may comprise substitution ofamino acid 109 of SEQ ID NO: 1 where any amino acid pre-existing atposition 109 is substituted. In certain embodiments, modified aminoacids are noted by their positions corresponding to the sequence in SEQID NO:1. For example, a native amino acid threonine at position 109 inSEQ ID NO:1 can be referred to as T109. In the foregoing example, nativeamino acid T109 can be substituted with the non-native amino acidtyrosine (Y), and this substitution is noted as T109Y. In certainembodiments, a portion of the alpha hemolysin polypeptide may betruncated. For example, the first 50 amino acids could be removed in analtered version of the protein pore, thus the protein pore would onlyhave 243 amino acids and positions rather than the normal 293. In thiscase, the modifications are still made in reference to the original SEQID NO:1. For example, in the truncated example, amino acid 59 wouldcorrespond to 109 in the original SEQ ID NO: 1. Thus, if native aminoacid 59 of the truncated protein pore was substituted with a non-nativeamino acid, it would still correspond to 109 of the original sequenceand be covered by this patent application. A native amino acid can be anamino acid found at a specific position within a sequence of a wild typehemolysin protein (e.g., the protein of SEQ ID NO: 1). The term “nativeamino acid substitution” as used herein, and/or any reference herein tosubstitution of a “native amino acid” refers to substitution orreplacement of a native amino acid with another amino acid that isdifferent than the native amino acid. A non-native amino acid can be astandard or non-standard amino acid that is different than a nativeamino acid found at a specific position within a sequence of a wild typehemolysin protein (e.g., the protein of SEQ ID NO: 1).

As used herein, the terms “aligned”, “alignment”, or “aligning” refer totwo or more nucleic acid sequences that can be identified as a match(e.g., 100% identity) or partial match. Alignments can be done manuallyor by a computer algorithm, examples including the Efficient LocalAlignment of Nucleotide Data (ELAND) computer program distributed aspart of the Illumina Genomics Analysis pipeline. The alignment of asequence read can be a 100% sequence match. In some cases, an alignmentis less than a 100% sequence match (i.e., non-perfect match, partialmatch, partial alignment). In some embodiments an alignment is about a99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89% 88%, 87%, 86%,85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76% or 75% match. In someembodiments, an alignment comprises a mismatch. In some embodiments, analignment comprises 1, 2, 3, 4 or 5 mismatches. Two or more sequencescan be aligned using either strand. In some cases a nucleic acidsequence is aligned with the reverse complement of another nucleic acidsequence.

An alignment often is used to determine sequence identity or homology.Sequence identity (e.g., percent sequence identity) and/or homology(e.g., percent homology) can be determined by any suitable alignmentprogram or algorithm. Percent sequence identity often refers to theamount of amino acids that match divided by the total amount of aminoacids aligned for two polypeptide sequences. Percent sequence homologyis often determined to compare two polypeptide sequences that maycomprises gap and/or inserts and often algorithms used for a homologydetermination weight amino acid alignments, in part, according toconservative and/or non-conservative substitutions.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTX and BLASTP, publicly available on theInternet at the NCBI website. Sequence searches are typically carriedout using the BLASTP program when evaluating a given amino acid sequencerelative to amino acid sequences in the Gen Bank Protein Sequences andother public databases. Both BLASTP and BLASTX can run using defaultparameters of an open gap penalty of 11.0, and an extended gap penaltyof 1.0, and utilize a BLOSUM-62 matrix, for example.

In certain embodiments, the modified alpha hemolysin amino acid sequencecomprises one or more native amino acid substitutions at one or morepositions corresponding to positions 1-109 and 149-293 of SEQ ID NO: 1.In certain embodiments, the modified alpha hemolysin amino acid sequencecomprises one or more native amino acid substitutions at one or morepositions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 97, 99, 101, 103, 105, 107, 109, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 225, 227, 229, 231 or 233 of SEQ IDNO: 1. In certain embodiments, the modified alpha hemolysin amino acidsequence comprises one or more native amino acid substitutions at one ormore positions corresponding to positions 105, 107, 109, 149, 151 or 153of SEQ ID NO: 1. In certain embodiments, the modified alpha hemolysinamino acid sequence comprises one or more native amino acidsubstitutions at one or more of positions corresponding to positions107, 109, 149, or 151 of SEQ ID NO: 1. In certain embodiments, themodified alpha hemolysin amino acid sequence comprises one or morenative amino acid substitutions at one or more positions correspondingto positions 109 or 149 of SEQ ID NO: 1.

In some embodiments herein, a polypeptide (e.g., a modified alphahemolysin protein) is an isolated polypeptide. An isolated polypeptidecan be isolated by a suitable isolation process known in the art. Apolypeptide or isolated polypeptide sometimes is provided as a purifiedpolypeptide, a partially purified polypeptide, an enriched polypeptide,an expressed polypeptide (e.g., an over-expressed polypeptide), asynthetic polypeptide (e.g., made by a chemical process, made by an invitro process) or the like. An expressed polypeptide can be expressed byany suitable method. An expressed polypeptide can be expressed in vitro(e.g., by an in vitro transcription and/or translation system), in situand/or in vivo by any suitable method. For example an expressedpolypeptide can be expressed using a suitable cell expression method orsystem. An isolated protein can be in the form of a lysate (cell, phageor bacterial lysate or virus lysate), cell or nuclear extract and/or asecretion product (e.g., in the form of conditioned media or spentbroth).

The term “isolated” as used herein refers to polypeptide (e.g., aprotein, or portion thereof) removed from its original environment(e.g., the natural environment if it is naturally occurring, or a hostcell if expressed exogenously), and thus is altered by humanintervention (e.g., “by the hand of man”) from its original environment.An isolated polypeptide can be a polypeptide removed from a subject(e.g., a human subject). An isolated polypeptide can be provided withfewer non-polypeptide components (e.g., nucleic acids, lipids,carbohydrates) than the amount of components present in a source sample.An isolated polypeptide can be provided with fewer contaminatingpolypeptide components (e.g., where contaminating polypeptides aredifferent polypeptides than the polypeptide intended to be isolated,e.g., different than the isolated polypeptide) than the amount ofcontaminating components present in a source sample. A compositioncomprising an isolated polypeptide can be about 50% to greater than 99%free of non-polypeptide components and/or contaminating polypeptidecomponents. A composition comprising an isolated polypeptide can beabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than99% free of non-polypeptide components and/or contaminating polypeptidecomponents. A composition comprising an isolated polypeptide cancontains fewer polypeptide species than in the sample source from whichthe polypeptide is derived. A composition comprising an isolatedpolypeptide may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% free of other polypeptide species. An isolatedpolypeptide can be provided in a mixture of polypeptides species (e.g.,an expression extract) where the isolated polypeptide comprises greaterthan 5%, 10%, 20%, 30%, 40%, 50% of the total protein content of themixture. For example, an isolated protein can be generated in an invitro translation expression system and can be used in a nanopore devicewith or without further purification.

The term “purified” as used herein can refer to a polypeptide providedthat contains fewer non-polypeptide components and/or contaminatingpolypeptide components (e.g., where contaminating polypeptides aredifferent polypeptides than the polypeptide intended to be purified,e.g., different than the purified polypeptide) than the amount ofnon-polypeptide components and/or contaminating polypeptide componentspresent prior to subjecting the polypeptide to a purification procedure.A composition comprising purified nucleic acid may be about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% free of non-polypeptidecomponents and/or contaminating polypeptide components. The term“purified” as used herein can refer to a polypeptide provided thatcontains fewer polypeptide species than in the sample source from whichthe polypeptide is derived. A composition comprising purifiedpolypeptide may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% free of other polypeptide species. For example,an exogenously expressed protein can be purified from a mixturecomprising endogenously expressed portions.

Types of Non-Native Amino Acid Substitutions

In some embodiments, the at least one native amino acid substituted witha non-native amino acid is substituted with a non-native amino acid thatis hydrophobic. In some embodiments, the at least one native amino acidsubstituted with a non-native amino acid is substituted with anon-native amino acid that is aromatic. In some embodiments, the atleast one native amino acid substituted with a non-native amino acid issubstituted with a non-native amino acid that is hydrophobic andaromatic. In some embodiments, the at least one native amino acidsubstituted with a non-native amino acid is substituted with anon-native amino acid that is larger than the native amino acid.

In certain embodiments, the non-native amino acid is naturallyoccurring. A naturally occurring amino acid can be any suitable standardamino acid or non-standard amino acid that is found in nature and can befound in a naturally occurring polymer (e.g., a naturally occurringpolypeptide or polynucleotide). In certain embodiments, a non-nativeamino acid is synthetic. In certain embodiments, the non-native aminoacid is hydrophobic and is methionine (M). In certain embodiments, thenon-native amino acid is hydrophobic and aromatic and is phenylalanine(F), tryptophan (W) or tyrosine (Y).

In certain embodiments, at least two native amino acids are substitutedwith non-native amino acids that are independently selected ashydrophobic, aromatic or hydrophobic and aromatic. For example, thenative amino acid threonine at position 109 (T109) is substituted withthe hydrophobic and aromatic non-native amino acid tyrosine (Y), thusT109Y while the native amino acid valine (V) at position 149 (V149) issubstituted with the hydrophobic amino acid methionine (M), thus V149M.In certain embodiments, at least three native amino acids aresubstituted with non-native amino acids that are independently selectedas hydrophobic, aromatic or hydrophobic and aromatic. In certainembodiments, at least four native amino acids are substituted withnon-native amino acids that are independently selected as hydrophobic,aromatic or hydrophobic and aromatic. In certain embodiments, at leastfive native amino acids are substituted with non-native amino acids thatare independently selected as hydrophobic, aromatic or hydrophobic andaromatic. In certain embodiments, at least one of the native amino acidsare substituted with non-native amino acids that are independentlyselected as hydrophobic, aromatic or hydrophobic and aromatic. In someembodiments, at least one of the native amino acids is substituted witha non-native amino acid that is not hydrophobic, aromatic or hydrophobicand aromatic as long as at least one of the native amino acids issubstituted with a non-native amino acid that is hydrophobic, aromaticor hydrophobic and aromatic.

In certain embodiments, the at least two native amino acids substitutedwith non-native amino acids are substituted with the same non-nativeamino acid. For example, native amino acids T109 and V149 are bothsubstituted with the non-native amino acid Y. In certain embodiments,the at least two native amino acids substituted with non-native aminoacids are substituted with non-native amino acids that are not the same.For example, native amino acid isoleucine (I) at position 107 (I107) issubstituted with the non-native amino acid tryptophan (W) while thenative amino acid V149 is substituted with the non-native amino acid Y,thus noted as I107W and V149Y.

In certain embodiments, the at least three native amino acidssubstituted with non-native amino acids are substituted with non-nativeamino acids that are not the same. For example, native amino acids I107,T109 and V149 are substituted with the non-native amino acids, M, W andY respectively. In certain embodiments, at least two of the at leastthree native amino acids substituted with non-native amino acids aresubstituted with the same non-native amino acid. For example, nativeamino acids I107, T109 and V149 are substituted with the non-nativeamino acids, W, W and Y respectively.

In some embodiments a substitution is a conservative substitution.Conservative amino acid substitutions are known in the art. Aconservative substitution often comprises substitution of a first aminoacid with a different second amino acid where the first and second aminoacids comprise similar physical properties (e.g., charge, size,hydrophobicity, the like). Non-limiting examples of conservativesubstitutions include replacing a hydrophobic amino acid (e.g., leucine)with different hydrophobic amino acid (e.g., valine), replacing a basicamino (e.g., lysine) with a different basic amino acid (e.g., arginine),and replacing a small flexible amino acid (e.g., glycine) with anothersmall flexible amino acid (e.g., serine) or the like. In someembodiments a substitution is a non-conservative substitution where afirst amino acid is substituted with another second amino acid where thefirst and second amino acids comprise substantially different physicalproperties.

Beta Barrel Amino Acids

In certain embodiments, a modified alpha hemolysin amino acid sequencecomprises one or more amino substitutions located in a beta barrel(e.g., in a beta barrel region). In certain embodiments, a substitutionin a beta barrel is chosen from one or more of positions 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145 and 147 of SEQ ID NO: 1. An amino acid located in a beta barrel isoften referred to herein as a beta barrel amino acid. In certainembodiments, at least two, at least three, at least four, at least five,at least six, at least seven or at least eight beta barrel amino acidsof a modified alpha hemolysin polypeptide are substituted (e.g.,replaced) with different amino acids. In some embodiments a modifiedalpha hemolysin protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18 or 19 amino acid substitutions located in a betabarrel. For example, the native amino acid T109 of wild type alphahemolysin is sometimes substituted with a non-native amino acid Y andthe beta barrel amino acid glutamic acid (E) at position 111 is replacedwith the different amino acid serine (S), thus termed T109Y E111 S. Incertain embodiments, the different amino acid is a naturally occurringamino acid. In certain embodiments, the different amino acid is asynthetic amino acid.

In certain embodiments, a polypeptide comprising a modified alphahemolysin amino acid sequence comprises the native amino acids T109 andV149 substituted with the non-native amino acid tyrosine (T109Y andV149Y) and the beta barrel amino acids E111, M113, D127, L135, T145 andK147 replaced with the different amino acids serine, serine, lysine,isoleucine, serine and serine respectively (E111S, M113S, D127K, L135I,T145S, and K147S), SEQ ID NO: 2.

In certain embodiments, a polypeptide comprising a modified alphahemolysin amino acid sequence comprises the native amino acids T109 andV149 substituted with the non-native amino acids tyrosine and tryptophan(W) respectively (T109Y and V149W) and the beta barrel amino acids E111,M113, D127, L135, T145 and K147 replaced with the different amino acidsserine, serine, lysine, isoleucine, serine and serine respectively (E111S, M113S, D127K, L135I, T145S, and K147S).

In some embodiments, a polypeptide comprising a modified alpha hemolysinamino acid sequence comprises the native amino acids T109 and V149substituted with the non-native amino acid tyrosine (T109Y and V149Y)and the beta barrel amino acids E111, M113, L135, T145 and K147 replacedwith the different amino acids serine, serine, isoleucine, serine andserine respectively (E111S, M113S, L135I, T145S, and K147S), SEQ ID NO:3.

In certain embodiments, a polypeptide comprising a modified alphahemolysin amino acid sequence comprises the native amino acids T109 andV149 substituted with the non-native amino acids tyrosine and tryptophan(W) respectively (T109Y and V149W) and the beta barrel amino acids E111,M113, D127, L135, T145 and K147 replaced with the different amino acidsserine, serine, isoleucine, serine and serine respectively (E111S,M113S, L135I, T145S, and K147S).

Polymer

A polymer, as referred to herein, can be any molecular polymer. Somecommon molecular polymers are polynucleotides and polypeptides. Apolymer can be a nucleic acid polymer, a protein polymer or a peptidepolymer. A polymer can be a single stranded or double stranded nucleicacid. A polymer can be a single stranded or double stranded DNA or RNA.A polymer can be a protein or peptide. Non-limiting examples of apolymer include a single stranded DNA, a double stranded DNA, a singlestranded RNA, a double stranded RNA, a protein and a peptide. In certainembodiments, the polymer is single stranded DNA. In certain embodiments,the polymer is double stranded DNA. A polymer can include one or moresections and a polymer section can include at least a portion of amonomer.

A monomer, as referred to herein, can be any molecule that can be linkedchemically to another molecule to form a polymer. A monomer can be anucleic acid or amino acid, for example. A monomer can be naturallyoccurring, modified or synthetic. A synthetic monomer is often a monomerthat is not found in a naturally occurring polymer. A naturallyoccurring polymer is often generated in nature, in vivo, by anun-altered or un-modified organism. In some embodiments a syntheticmonomer is a synthetic amino acid or synthetic nucleotide. A nucleicacid monomer can be phosphorylated, oxidized, acetylated, methylated orsulfonated. A nucleic acid monomer can be a monophosphate nucleotide,modified nucleotide, methylated nucleotide, acetylated nucleotide oroxidized nucleotide. Non-limiting examples of a monomer includenucleotides, monophosphate nucleotides, modified nucleotides, methylatednucleotides and oxidized nucleotides. Non-limiting examples of a nucleicacid monomer include adenine (A), cytosine (C), thymine (T), guanine(G), uracil (U), modified cytosine, 7-methylguanine, xanthine,hypoxanthine, 5,6-dihydrouracil, 5-methylcytosine, N4-methylcytosine,hydroxymethylcytosine, or N6-methyladenine.

A single stranded polynucleotide consisting of a certain nucleotidemonomer (N) is referred to herein as polyN, polyNi or polyNiNi where iindicates the number of monomers in a polynucleotide. For example, asingle stranded polynucleotide consisting of adenine monomers can bereferred to herein as a polyA, a polynucleotide of cytosine monomers canbe referred to herein as polyC, a polynucleotide of thymine monomers canbe referred to herein as polyT, and a polynucleotide of guanine monomerscan be referred to as polyG. For example, a polymer that is singlestranded DNA consisting of 100 cytosine (C) bases is referred to hereinas polyC100 (SEQ ID NO: 4). A polymer that is single stranded DNAconsisting of 50 cytosine bases followed by 50 adenine (A) bases isreferred to herein as polyC50A50 (SEQ ID NO: 6), for example.

Polymer Slowing

In certain embodiments, a polypeptide comprising a modified alphahemolysin amino acid sequence that comprises at least one amino acidsubstituted with a non-native amino acid forms a modified alphahemolysin polypeptide. In certain embodiments, a modified alphahemolysin polypeptide enables a polymer to at least partiallytranslocate through the modified alpha hemolysin polypeptide. In certainembodiments, the translocation time of a polymer through a modifiedalpha hemolysin polypeptide is increased relative to the translocationtime of the same polymer through a reference alpha hemolysinpolypeptide.

A reference alpha hemolysin polypeptide sometimes is modified relativeto a wild type counterpart of the pore, and often the reference alphahemolysin polypeptide is an unmodified wild type protein pore. Areference alpha hemolysin polypeptide can be an unmodified or modifiedwild type alpha hemolysin polypeptide. In some embodiments a referencealpha hemolysin polypeptide comprises an amino acid sequence comprisingat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99%identity to SEQ ID NO: 1. In some embodiments a reference alphahemolysin polypeptide consists of the amino acid sequence of SEQ ID NO:1.

In some embodiments a reference alpha hemolysin polypeptide comprises anamino acid sequence comprising at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In some embodiments areference alpha hemolysin polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14,15, 16,17, 18, 19 or 20 or more amino acidsubstitutions relative to SEQ ID NO: 1. In some embodiments a referencealpha hemolysin protein comprises an alpha hemolysin amino acid sequencecomprising one or more amino acid modifications in addition to one ormore native amino acid substitutions described herein for a modifiedalpha hemolysin polypeptide. In certain embodiments, a reference alphahemolysin protein comprises one or more amino acid substitutions,deletions or insertions in a beta barrel region of an alpha hemolysinprotein. In some embodiments a reference alpha hemolysin polypeptidecomprises one or more amino acid substitution at one or more positionscorresponding to positions 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145 or 147 of SEQ ID NO: 1. Insome embodiments a reference alpha hemolysin polypeptide does notcomprise an amino acid substitution at one or more positionscorresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 97, 99, 101, 103, 105, 107, 109, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 225, 227, 229, 231 or 233 of SEQ ID NO: 1.In some embodiments a reference alpha hemolysin polypeptide does notcomprise an amino acid substitution at one or more positionscorresponding to positions 105, 107, 109, 149, 151 or 153 of SEQ IDNO: 1. In some embodiments a reference alpha hemolysin polypeptide doesnot comprise an amino acid substitution at one or more positionscorresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 97, 99, 101, 103, 105, 107, 109, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 225, 227, 229, 231 or 233 of SEQ ID NO: 1,where the amino acid substitution independently is to a non-nativehydrophobic amino acid or non-native aromatic amino acid, or non-nativearomatic and hydrophobic amino acid.

A translocation time of a polymer (e.g., a polynucleotide, apolypeptide) through a modified alpha hemolysin polypeptide can bedetermined (e.g., measured) and compared to a translocation time of thesame polymer through another alpha hemolysin polypeptide (e.g., a wildtype alpha hemolysin polypeptide, a reference alpha hemolysinpolypeptide). For example, a translocation time for a wild type alphahemolysin polypeptide, such as an alpha hemolysin polypeptide having theamino acid sequence of SEQ ID NO: 1, is compared to the translocationtime of a modified alpha hemolysin polypeptide counterpart (e.g., amodified alpha hemolysin polypeptide described herein. In someembodiments a polymer translocates through a modified alpha hemolysinpolypeptide at a slower rate than the same polymer translocates througha wild type alpha hemolysin polypeptide. The time required for a polymerto translocate through an alpha hemolysin polypeptide is referred toherein as a translocation time. A polymer that translocates through afirst pore at a slower rate than a second pore often comprises a longertranslocation time through the first pore when compared to the secondpore. In some embodiments a polymer translocates through a modifiedalpha hemolysin polypeptide with a longer translocation time whencompared to a translocation time of the same polymer through a referencepore (e.g., a wild type alpha hemolysin polypeptide).

In certain embodiments, a modified alpha hemolysin polypeptidecomprising one or more amino acid substitutions outside of a beta barrelregion enables slowing (e.g., increases the translocation time) of apolymer compared to a reference pore. In certain embodiments a modifiedalpha hemolysin polypeptide and a reference pore to which it is comparedboth comprise one or more amino acid substitutions within a beta barrelregion. For example, a reference pore used for comparison sometimes doesnot have amino acid substitutions outside of a barrel region, but doescomprise one or more beta barrel amino acid substitutions. For example,as described herein, the translocation time of the polymer polyC100 (SEQID NO: 4) is increased in the modified alpha hemolysin polypeptide T109YE111S M113S L135S T145S K147S V149Y compared to the alpha hemolysinpolypeptide E111S M113S L135S T145S K147S that, as described herein,does not have a an amino acid substitution outside of a barrel region,but does comprise beta barrel amino acid substitutions.

The presence or absence of a change (e.g., a relative change; anincrease) in the translocation time for the modified alpha hemolysinpolypeptide, relative to the translocation time of the same polymerthrough a reference alpha hemolysin polypeptide, often is determined(e.g., measured) and/or quantified. A translocation time can bedetermined using a polyC100 (SEQ ID NO: 4) or polyA100 (SEQ ID NO: 5)polymer for the modified protein and the reference protein. A testingprocedure that can be used for assessing translocation time includesdetermining the translocation time of a polyC100 (SEQ ID NO: 4) orpolyA100 (SEQ ID NO: 5) polymer for the modified protein and thereference protein, where the reference protein is an alpha hemolysinpolypeptide having the amino acid sequence of SEQ ID NO: 1. Detailedmethods for determining (e.g., measuring) the translocation time of apolymer (e.g., a polynucleotide, a polypeptide) through an alphahemolysin polypeptide are described herein. In certain embodiments, anincrease in a translocation time of a polymer through a modified alphahemolysin polypeptide is at least about 20 percent (e.g. at least about30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,1000 or 2000 percent) compared to the translocation time of the samepolymer through an alpha hemolysin polypeptide without at least onenative amino acid substituted with the non-native amino acid (e.g., awild type alpha hemolysin polypeptide).

Improvements as a Result of Polymer Slowing

In certain embodiments, the modified alpha hemolysin polypeptide can beused for sequencing a polymer. In certain embodiments, the slowing of apolymer enabled by the modified alpha hemolysin polypeptide can improvethe accuracy of sequencing the polymer. For example, if a polymer has anincreased translocation time through a protein pore, then a lowermeasurement bandwidth can be used. The lower measurement bandwidthresults in lower measurement noise, thus increasing the signal to noiseratio. In the case of sequencing, the signal to noise ratio can betermed contrast signal to noise ratio (CNR) to demonstrate thedifference between two bases (e.g. the signal difference between adenineand cytosine) compared to the noise. This concept and protein pores withhigh CNR values are described in patent application nos.PCT/US2012/04859 and PCT/US2012/04864, both herein incorporated byreference.

In certain embodiments, the modified alpha hemolysin polypeptide permitsa measurement of a first level and a second level within a residualcurrent of the modified alpha hemolysin polypeptide, as the polymertranslocates through the modified alpha hemolysin polypeptide, with acontrast signal to noise ratio (CNR) computed at a predetermined filterfrequency. In certain embodiments, the polymer comprises two or moresections, each of which sections comprises at least a portion of amonomer. The CNR is calculated as a contrast signal divided by a noisevalue where the contrast signal is calculated as the difference betweenthe first level and the second level and each level used for calculatingthe CNR correlates to a composition of a section of the polymer.Furthermore, the first level and the second level are measurablydistinct. The noise value is computed at the predetermined filterfrequency. In certain embodiments, the increase in translocation time ofthe polymer enables a lower predetermined filter frequency to be used inthe computation of the CNR. In certain embodiments, the lowerpredetermined frequency is at least 5 percent lower (e.g. at least 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent lower). In someembodiments, the lower predetermined filter frequency results in a lowernoise value. In certain embodiments, the lower noise value is at least 5percent lower (e.g. at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95percent lower). In some embodiments, the lower noise value results in ahigher CNR. In certain embodiments, the higher CNR is at least 5 percenthigher (e.g. at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 1000 percent higher). In some embodiments, a higher CNR enablessequencing of the polymer. In certain embodiments, a higher CNR enablesmore accurate sequencing of the polymer.

In some embodiments, the CNR of the modified alpha hemolysin proteinpore is lower than the CNR for the protein pore without the at least onenative amino acid substituted with the non-native amino acid at the samepredetermined filter frequency. In certain embodiments, the lowerpredetermined filter frequency that can be used as a result of theincrease in translocation time results in a lower noise value. Incertain embodiments, the lower noise value results in a higher CNR atthe lower predetermined filter frequency for the modified alphahemolysin protein pore than the CNR for the protein pore without the atleast one native amino acid substituted with the non-native amino acidat the higher predetermined filter frequency. In certain embodiments,the higher CNR enables sequencing of the polymer. In certainembodiments, the higher CNR enables more accurate sequencing of thepolymer. In certain embodiments, the noise associated with the polymerin the modified alpha hemolysin protein pore and/or the protein porewithout the at least one native amino acid substituted with thenon-native amino acid is white noise (i.e. has a relatively flat powerspectral density) in the bandwidth of interest (e.g. 100 to 20,000 Hz).In certain embodiments, the higher CNR at the lower predetermined filterfrequency can be estimated when the noise associated with the polymer inthe modified alpha hemolysin protein pore and/or the protein porewithout the at least one native amino acid substituted with thenon-native amino acid is white noise. In some embodiments, when thenoise is white noise, the noise at the lower predetermined filterfrequency (PDL) is equal to the noise at the higher predetermined filterfrequency (PDH) multiplied by the square root of the PDL divided by thePDH. For example, the noise at the PDL of 1000 Hz is equal to the noiseat the PDH of 4000 Hz multiplied by the square root of 1000/4000. Incertain embodiments, when the noise is white noise, if the tmax isincreased, then the predetermined filter frequency can be reduced by thesame amount the tmax is increased. For example, if the tmax is increasedby a factor of 2, then the predetermined filter frequency can be reducedby a factor of 2. In certain embodiments, a Weighted CNR, due to thedescribed relationship between tmax and the bandwidth, can be estimatedby multiplying the CNR at the predetermined filter frequency by thesquare root of the tmax. In some embodiments, a ratio of the WeightedCNR of the modified alpha hemolysin protein pore to the Weighted CNR ofthe protein pore without the at least one native amino acid substitutedwith the non-native amino acid factor is computed. In certainembodiments, the ratio of Weighted CNRs is computed by dividing theWeighted CNR of the modified alpha hemolysin protein pore by theWeighted CNR of the protein pore without the at least one native aminoacid substituted with the non-native amino acid. In certain embodiments,if the ratio is greater than 1, then the modified alpha hemolysinprotein pore has a higher Weighted CNR than the protein pore without theat least one native amino acid substituted with the non-native aminoacid. In some embodiments, a higher Weighted CNR enables sequencing ofthe polymer. In certain embodiments, a higher Weighted CNR enables moreaccurate sequencing of the polymer.

Nanopore Devices

In some embodiments, provided is a device or apparatus, termed nanoporedevice, that includes a modified alpha hemolysin polypeptide describedherein. Any suitable device capable of supporting a modified alphahemolysin polypeptide and allowing for sensing of an analyte can beutilized. Nanopore devices are often comprised of a substrate thatincludes an aperture and one or more proteins or polypeptides insertedin the aperture. In certain embodiments, the protein is inserted in alipid monolayer and/or bilayer that traverses the aperture. In someembodiments, the protein is retained within the aperture without a lipidmonolayer and/or bilayer. In some embodiments, a substrate includes awell and one or more proteins inserted in the well opening within alipid monolayer and/or bilayer that traverses the well opening. Incertain embodiments, a substrate includes a well and one or moreproteins inserted in the well opening without a lipid monolayer and/orbilayer that traverses the well opening.

In certain embodiments, an apparatus or device comprising a hemolysinpolypeptide comprises a direct current (DC) measurement system. In someembodiments, an apparatus or device comprising a hemolysin polypeptidecomprises an alternating current (AC) measurement system. In certainembodiments, an apparatus or device comprises an AC/DC measurementsystem. Conditions in which a polymer translocates through a proteinpore (e.g., a hemolysin polypeptide, an apparatus or device comprising ahemolysin pore) often include an applied voltage bias. In someembodiments, for measuring DC potential in a nanopore system comprisinga hemolysin pore protein (e.g., for DC measurement systems), a voltagebias is applied across a pore to produce a measurable current. A voltagebias is often held constant for a measurement of a CNR. A voltage biasused in a nanopore residual current measurements can have an effect on ameasured CNR. In general, as a bias is increased, the contrast betweenpolymer sections increases and the average duration per polymer sectionwill decrease. Non-limiting examples of bias ranges that can be used tomeasure a CNR include 20 millivolts (mV) to 300 mV or greater (e.g., 20mV, 30 mV, 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, 90 mV, 100 mV, 110 mV, 120mV, 130 mV, 140 mV, 150 mV, 160 mV, 170 mV, 180 mV, 190 mV, 200 mV, 210mV, 220 mV, 230 mV, 240 mV, 250 mV, 260 mV, 270 mV, 280 mV, 290 mV, 300mV or greater). In some embodiments, for an AC measurement system, asource signal that is periodic (e.g. sinusoidal or a square wave) isapplied and is defined by an applied bias (e.g. an AC bias) andfrequency. Conditions in which a polymer translocates through a proteinpore (e.g., a hemolysin polypeptide) often include an applied AC bias.Non-limiting examples of an applied AC bias include 50 mV to 1000 mV(e.g. 50 mV, 60 mV, 70 mV, 80 mV, 90 mV, 100 mV, 110 mV, 120 mV, 130 mV,140 mV, 150 mV, 160 mV, 170 mV, 180 mV, 190 mV, 200 mV, 300 mV, 400 mV,500 mV, 600 mV, 700 mV, 800 mV, 900 mV, or 1000 mV). Non-limitingexamples of the frequency include 10 kHz to 300 kHz or greater (e.g. 10kHz, 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, 100kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz or greater).

In some embodiments, the substrate comprises glass, Si, SiO₂, Si₃ N₄ ,alumina, nitrides, diamond, quartz, sapphire metals, ceramics,alumino-silicate, polymers (e.g., Teflon, polycarbonate), the like orcombinations thereof. Non-limiting examples of glass types suitable fora substrate include fused silica glass, ninety-six percent silica glass,soda-lime silica glass, borosilicate glass, aluminosilicate glass, leadglass, doped glass comprising desired additives, functionalized glasscomprising desired reactive groups, the like and combinations thereof.Non-limiting examples of minerals (e.g., quartz) suitable for asubstrate include quartz, tridymite, cristobalite, coesite,lechatelierite, stishovite, the like and combinations thereof. Thesubstrate can be manufactured from a pure substance or can bemanufactured from a composite material.

The thickness of a substrate typically ranges from about 100 nanometer(nm) to 5 millimeters (mm) in thickness (e.g., about 100 nm, about 150nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900nm, about 1000 nm (e.g., about 1 μm), about 2 μm, about 3 μm, about 4μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm,about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about80 μm, about 90 μm, 100 μm, about 110 μm, about 120 μm, about 130 μm,about 140 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm,about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm,about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm,1000 μm (e.g. 1 mm), about 2 mm, about 3 mm, about, about 4 mm, or about5 mm).

In certain embodiments, a substrate contains an aperture that separatestwo fluid reservoirs. In some embodiments, the aperture is a micronscale aperture, and sometimes the aperture is a nanoscale aperture. Insome embodiments, the aperture is in a glass or quartz substrate. Incertain embodiments, the aperture has a diameter of about 0.25 nanometerto about 100 μm (e.g., about 0.25 nanometers, about 0.5 nanometers,about 1 nanometer, about 1.5 nanometers, about 2 nanometers, about 2.5nanometers, about 3 nanometers, about 3.5 nanometers, about 4nanometers, about 4.5 nanometers, about 5 nanometers, about 6nanometers, about 7 nanometers, about 8 nanometers, about 9 nanometers,about 10 nanometers, about 15 nanometers, about 20 nanometers, about 25nanometers, about 30 nanometers, about 35 nanometers, about 40nanometers, about 45 nanometers, about 50 nanometers, about 60nanometers, about 70 nanometers, about 80 nanometers, about 90nanometers, about 100 nanometers, about 125 nanometers, about 150nanometers, about 175 nanometers, about 200 nanometers, about 250nanometers, about 300 nanometers, about 350 nanometers, about 350nanometers, about 400 nanometers, about 500 nanometers, about 600nanometers, about 700 nanometers, about 800 nanometers, about 900nanometers, about 1000 nanometers (e.g., 1 μm), about 2 μm, about 3 μm,about 4 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, or about 50 μm).

In certain embodiments, a substrate comprises a well. In someembodiments, the well has an aperture formed by the well opening with adiameter of about 100 nanometers to about 100 μm (e.g., about 100nanometers, about 125 nanometers, about 150 nanometers, about 175nanometers, about 200 nanometers, about 250 nanometers, about 300nanometers, about 350 nanometers, about 350 nanometers, about 400nanometers, about 500 nanometers, about 600 nanometers, about 700nanometers, about 800 nanometers, about 900 nanometers, about 1000nanometers (e.g., 1 μm), about 2 μm, about 3 μm, about 4 μm, about 5 μm,about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm,about 80 μm, about 90 μm or about 100 μm).

The channel formed by the aperture in a substrate is of any suitablegeometry, and sometimes has a substantially circular, oval, square,rectangular, rhomboid, parallelogram, or other like cross-section. Thechannel in the substrate is of any suitable profile, and sometimes has asubstantially cylindrical or conical (e.g., tapering or expandingconical) profile.

A substrate sometimes comprises a coating that modifies the surface ofan aperture or well structure. In some embodiments, a substratecomprises a surface that includes a hydrophobic substance. In certainembodiments, a substrate comprises a surface that includes a hydrophilicsubstance. In some embodiments, a substrate comprises a surface thatincludes hydrophobic and hydrophilic substances.

Thus, one or more portions of, or the entire, substrate can be treatedor coated to adopt certain desirable characteristics, in someembodiments. In certain embodiments, the treatment or coating enhancesformation of lipid structures across the aperture of the substrate.Physical and/or chemical modification of the surface properties of asubstrate include, but are not limited to, modification of theelectrical charge density, changes to the hydrophobicity, changes to thehydrophilicity, the like and combinations thereof. Any suitablesubstance can be utilized to modify one or more interior and/or exteriorsurfaces of the substrate. Non-limiting examples of suitable materialsfor modification of one or more substrate surfaces include silanes,silanes terminating in a cyano group, silanes terminating in a methylgroup, thiols, the like, or combinations thereof. In some embodiments,an exterior surface of a substrate may be modified by a first entity. Incertain embodiments, an interior surface of a substrate may be modifiedby a second entity. In some embodiments, the first and the second entitymay be the same entities, and in certain embodiments, the first and thesecond entity may be different entities. In some embodiments utilizing aglass substrate, the first or second entities that can be used to modifythe interior or exterior surfaces of a substrate include a variety ofglass-reactive species, e.g., 3-cyano-propyldimethylchlorosilane, thatreact with the silanol groups of the glass surface.

In some embodiments, a device comprises a lipid composition (e.g.,monolayer, bilayer, combination thereof) over, across or spanning anaperture of a substrate. A lipid composition sometimes comprises a lipidmonolayer, sometimes comprises a lipid bilayer, and in some embodimentscomprises a lipid layer that partially is a monolayer and partially is abilayer. In some devices comprising both monolayer and bilayer lipidstructures, solvent may be trapped at a location (e.g., an annulus)between the substrate and the lipid layer at or near the monolayer andbilayer interface, which is addressed in greater detail hereafter.

The lipid composition of a device often is relatively stable tomechanical disturbances, and can have a lifetime in excess of two weeks.Additionally, a device can be made with a lipid composition that isreadily formed over or in an aperture and has a relatively small surfacearea, which can give rise to favorable electrical characteristics.

Nanopore membrane devices can comprise a channel or nanopore embedded ina suitable material. The diameter of an aperture of a channel in amembrane, across which an amphiphilic composition forms in a nanoporemembrane system, often ranges in diameter from about 0.25 nanometers toabout 50 μm (e.g., about 0.25 nanometers, about 0.5 nanometers, about 1nanometer, about 1.5 nanometers, about 2 nanometers, about 2.5nanometers, about 3 nanometers, about 3.5 nanometers, about 4nanometers, about 4.5 nanometers, about 5 nanometers, about 6nanometers, about 7 nanometers, about 8 nanometers, about 9 nanometers,about 10 nanometers, about 15 nanometers, about 20 nanometers, about 25nanometers, about 30 nanometers, about 35 nanometers, about 40nanometers, about 45 nanometers, about 50 nanometers, about 60nanometers, about 70 nanometers, about 80 nanometers, about 90nanometers, about 100 nanometers, about 125 nanometers, about 150nanometers, about 175 nanometers, about 200 nanometers, about 250nanometers, about 300 nanometers, about 350 nanometers, about 350nanometers, about 400 nanometers, about 500 nanometers, about 600nanometers, about 700 nanometers, about 800 nanometers, about 900nanometers, about 1000 nanometers (e.g., 1 μm), about 1.5 μm, about 2μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 5 μm,about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about35 μm, about 40 μm, about 45 μm, or about 50 μm). The channel formed inthe membrane is of any suitable geometry, and sometimes has asubstantially circular, oval, square, rectangular, rhomboid,parallelogram, or other like cross-section. The channel formed in themembrane is of any suitable profile, and sometimes has a substantiallycylindrical or conical (e.g., tapering or expanding conical) profile.Nanopore membrane devices often are composed of a single conical-shapedchannel or nanopore embedded in a suitable material. Membranes can beformed as known in the art and as described herein.

While a device often comprises a lipid composition traversing asubstrate aperture, the composition traversing the substrate aperturemay comprise any suitable amphiphilic molecule(s) or material(s) thatcan stably traverse an aperture and into which a protein can beincorporated. An amphiphilic molecule generally is composed of ahydrophobic portion and a polar portion. The terms “amphiphilicmaterial” or “amphiphilic materials” refer to materials made ofmolecules having a polar, water-soluble group attached to a nonpolar,water-insoluble hydrocarbon chain. Amphiphilic materials sometimes canbe polymers. Amphiphilic materials may be a pure substance or a mixtureof different amphiphilic materials. The polymeric materials may be apolymer with a uniform molecular weight distribution, or a polymer witha non-uniform molecular weight distribution, or a mixture of polymerswhich comprise different monomers. Non-limiting examples of amphiphilicmaterials include lipids, detergents, surfactants, proteins,polysaccharides, and other chemical or biochemical materials that can berendered amphiphilic.

The terms “detergent” or “detergents” as used herein refer to asurfactant or a mixture of surfactants. In some embodiments,“surfactant” or “surfactants” refer to any compound that (i) lowers thesurface tension of a liquid, allowing easier spreading, and/or (ii)lowers the interfacial tension between two liquids, or between a liquidand a solid. Surfactants may act as: detergents, wetting agents,emulsifiers, foaming agents, and dispersants. Surfactants often arecategorized as ionic (anionic or cationic), zwitterionic or amphoteric,or non-ionic. Non-limiting examples of surfactants include ammoniumlauryl sulfate, sodium lauryl sulfate (SDS), sodium laureth sulfate(e.g., also known as sodium lauryl ether sulfate (SLES)), sodium myrethsulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate (PFOS),perfluorobutanesulfonate, alkyl benzene sulfonates, alkyl aryl etherphosphate, alkyl ether phosphate, fatty acid salts (e.g., soaps), sodiumstearate, sodium lauroyl sarcosinate, perfluorononanoate,perfluorooctanoate, octenidine dihydrochloride, cetyl trimethylammoniumbromide (CTAB), cetyl trimethylammonium chloride (CTAC), Cetylpyridiniumchloride (CPC), polyethoxylated tallow amine (POEA), benzalkoniumchloride (BAC), benzethonium chloride (BZT);5-Bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride,dioctadecyldimethylammonium bromide,3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (e.g., CHAPS),cocamidopropyl hydroxysultaine, amino acids, imino acids, cocamidopropylbetaine, lecithin, fatty alcohols (e.g., cetyl alcohol, stearyl alcohol,and the like), the like and combinations thereof.

A lipid molecule typically comprises at least one hydrophobic chain andat least one polar head. When exposed to an aqueous environment, lipidsoften will self-assemble into structures that minimize the surface areaexposed to a polar (e.g., aqueous) medium. Lipids sometimes assembleinto structures having a single or monolayer of lipid enclosing anon-aqueous environment, and lipids sometimes assemble into structurescomprising a bilayer enclosing an aqueous environment. In a monolayerstructure, the polar portion of lipids (e.g., the head of the moleculein the case of phospholipids and other lipids commonly found in cellsubstrates) often is oriented towards the polar, aqueous environment,allowing the non-polar portion of the lipid to contact the non-polarenvironment.

A lipid bilayer typically comprises a sheet of lipids, generally twomolecules thick, arranged so the hydrophilic phosphate heads pointtowards a hydrophilic aqueous environment on either side of the bilayerand the hydrophobic tails point towards the hydrophobic core of thebilayer. This arrangement results in two “leaflets” that are each asingle molecular layer. Lipids self-assemble into a bilayer structuredue to the hydrophobic effect and are held together entirely bynon-covalent forces that do not involve formation of chemical bondsbetween individual molecules. Lipid bilayers generally also areimpermeable to ions, which allow cells to regulate various processesthat involve salt concentrations or gradients and intracellular pH bypumping ions across cell substrates using ion transport mechanisms.

In some embodiments, lipid bilayers are natural, and in certainembodiments lipid bilayers are artificially generated. Natural bilayersoften are made mostly of phospholipids, which have a hydrophilic headand two hydrophobic tails (e.g., lipid tails), and form a two-layeredsheet as noted above, when exposed to water or an aqueous environment.The center of this bilayer contains almost no water and also excludesmolecules like sugars or salts that dissolve in water, but not in oil.Lipid tails also can affect lipid composition properties, by determiningthe phase of the bilayers, for example. A bilayer sometimes adopts asolid gel phase state at lower temperatures and undergoes a phasetransition to a fluid state at higher temperatures. The packing oflipids within a bilayer also affects its mechanical properties,including its resistance to stretching and bending.

Artificial bilayers (e.g., sometimes also referred to as “model lipidbilayers”) are any bilayers assembled through artificial means, asopposed to bilayers that occur naturally (e.g., cell walls, lipidbilayers that cover various sub-cellular structures). An artificialbilayer can be made with synthetic and/or natural lipids, thus theprocess, not the material, defines an artificial or model system.Properties, such as stretching, bending or temperature induced phasetransitions, have been studied with artificial model bilayers. Thesimplest model systems contain only a single pure synthetic lipid. Theartificial bilayer also may contain a hydrophobic solvent, such asdecane, hexadecane, pentane or other solvents and combinations thereof,that is used to disperse the lipid during bilayer formation andstabilize the formation of lipid bilayers across apertures inhydrophobic materials. The simplicity of a single lipid system isadvantageous when determining physical or mechanical properties ofbilayers. Model bilayers with greater physiological relevance can begenerated utilizing mixtures of several synthetic lipids or, asmentioned, with natural lipids extracted from biological samples.

The presence of certain lipids or proteins sometimes can alter thesurface chemistry of bilayers (e.g., viscosity or fluidity of lipidbilayers). Phospholipids with certain head groups can alter the surfacechemistry of a bilayer. Non-limiting examples of bilayer constituentsthat can alter the surface chemistry of bilayers include fats, lecithin,cholesterol, proteins, phospholipids (e.g., phosphatidic acid(phosphatidate), phosphatidylethanolamine (e.g., cephalin),phosphatidylcholine (e.g., lecithin), phosphatidylserine, andphosphoinositides such as phosphatidylinositol (PI),phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate(PIP2) and phosphatidylinositol triphosphate (PIP3),phosphatidylglycerol, ceramide phosphorylcholine, ceramidephosphorylethanolamine, ceramide phosphorylglycerol), surfactants, thelike and combinations thereof.

A device may include one or more types of molecules other thanphospholipids. For example, cholesterol, which helps strengthen bilayersand decreases bilayer permeability can be included. Cholesterol alsohelps regulate the activity of certain integral substrate proteins.Different types or forms of lipid compositions (e.g., monolayers and/orbilayers) can be found naturally or generated artificially. Non-limitingexamples of lipid compositions include monolayers (e.g., micelles) andbilayers including “black PLBs”, vesicles (e.g., sometimes referred toas “liposomes”), supported lipid bilayers, linear lipid bilayers and thelike.

A nanopore membrane device often comprises a lipid composition (e.g.,monolayer, bilayer, combination thereof) over, across or spanning anaperture of a substrate. A lipid composition can comprise one or moretypes of lipids having various chain lengths and/or various structuresof polar heads. A lipid composition of a nanopore membrane device oftenis relatively stable to mechanical disturbances, and can have a lifetimein excess of two weeks. A lipid composition sometimes comprises a lipidmonolayer, sometimes comprises a lipid bilayer, and in some embodimentscomprises a lipid layer that partially is a monolayer and partially is abilayer. A portion of a lipid composition in a device can interact withone or more exterior and/or interior surfaces of a substrate. In somedevices comprising both monolayer and bilayer lipid structures, solventmay be trapped at a location (i.e., annulus) between the substrate andthe lipid layer at or near the monolayer and bilayer interface, which isaddressed in greater detail hereafter. In certain embodiments, a lipidcomposition that spans across the substrate aperture is a combination ofa lipid bilayer and monolayer. In various embodiments, a lipid monolayerdeposited on the exterior surface of a substrate and a lipid monolayerdeposited on the interior surface of the channel or nanopore that jointogether at about the edge of the channel or nanopore opening can form alipid bilayer spanning or suspended across the aperture. The bilayerformed across an aperture sometimes is referred to as a “spanning lipidbilayer” herein.

In a spanning bilayer structure, a bilayer often is present across thesubstrate aperture and a monolayer is present on substrate surfaces(e.g., chemically modified surfaces and/or hydrophobic). In someembodiments, a chemically modified device corrals a single protein porein the lipid bilayer region that spans across the aperture. An insertedprotein (e.g., protein pore, alpha hemolysin, modified alpha hemolysinpolypeptide) often is able to diffuse in the bilayer across the aperturebut often cannot leave this area to enter the lipid monolayer. Insertionof a sensing entity (e.g., protein pore) often occurs only in thebilayer region. A thin layer (e.g., about 1 to about 10 nm thick)containing solvent and ions sometimes is formed between a spanning lipidbilayer and one or more surfaces of the substrate. The thickness of thislayer is defined as the distance between the exterior surface and thelipid bilayer and often plays a role in determining the resistance ofthe bilayer seal and the stability and fluidity of the bilayer. Aspanning bilayer also sometimes includes an annulus formed betweenmonolayers and a channel or nanopore surface, which can contain solvent(e.g., FIG. 15 of U.S. Pat. No. 7,777,505).

A protein often is inserted into a structure (e.g., monolayer and/orbilayer) formed by the lipid or amphiphilic material composition. Aprotein that is inserted into the structure can be water soluble,detergent-solubilized or incorporated into a lipid bilayer (e.g.,vesicle, liposome) or a lipid monolayer (e.g., micelle) prior toinsertion into a PLB, in some embodiments. Membrane proteins sometimescannot be incorporated directly into the PLB during formation becauseimmersion in an organic solvent sometimes can denature the protein.Exceptions include alpha hemolysin, MspA, and gramicidin. A membraneprotein sometimes is solubilized with a detergent and added to theaqueous solution after the bilayer is formed. The dilution of thedetergent stabilizing the protein forces the proteins to spontaneouslyinsert into the bilayer over a period of minutes or hours, and often ata low frequency of success.

A vesicle is a lipid bilayer configured as a spherical shell enclosing asmall amount of water or aqueous solution and separating it from thewater or aqueous solution outside the vesicle. Because of thefundamental similarity to a cell wall, vesicles have been used to studythe properties of lipid bilayers. Vesicles also are readilymanufactured. A sample of dehydrated lipid spontaneously forms vesicles,when exposed to water. Spontaneously formed vesicles can be unilamelar(single-walled) or multilamellar (e.g., many-walled) and are of a widerange of sizes from tens of nanometers to several micrometers. Aliposome is an artificially prepared vesicle, and also comprises a lipidbilayer and also can be made of naturally occurring or synthetic lipids,including phospholipids. There are four types of liposomes: MLV(multilamellar vesicles), SUV (Small Unilamellar Vesicles), LUV (LargeUnilamellar Vesicles) and GUV (Giant Unilamellar Vesicles). Liposomesmay be used to form PLBs on a surface or across apertures.

Unlike a vesicle or a cell substrate in which the lipid bilayer forms anenclosed shell, a supported bilayer (e.g., SLB) is a planar structure incontact with a substrate. One advantage of the supported bilayer is itsstability. SLBs often remain largely intact even when subject to highflow rates or vibration, and the presence of holes will not destroy theentire bilayer. Due to the stability of SLB's, experiments lasting weeksand even months can be conducted with supported bilayers, while BLMexperiments sometimes are limited to hours. Another advantage of thesupported bilayer is the greater number of methods and tools useable forcharacterization. In certain embodiments, a substrate may comprise ahydrophilic material, such as untreated glass, or it may be modified ina manner that renders one or more surfaces of the substrate (e.g., poreinterior, pore exterior) hydrophilic (e.g. mildly hydrophilic,substantially hydrophilic). In certain embodiments, the bilayer is thenformed over the hydrophilic surface and covers across the substrateaperture.

In certain embodiments, a substrate may include a hydrophobic material,such as Teflon, or it may be modified in a manner that renders one ormore surfaces of the substrate (e.g., substrate channel interior,substrate channel exterior) hydrophobic (e.g. mildly hydrophobic,substantially hydrophobic). In some embodiments one or more surfaces ofa substrate are coated with a hydrophobic substance, including withoutlimitation an alkyl silane substance (e.g.,3-cyano-propyldimethylchlorosilane). Any suitable silane substance canbe selected to render a substrate surface more hydrophobic and supportinteraction with lipids for formation of a lipid structure that spansthe substrate aperture. In some embodiments, a spanning lipid structurecontains a monolayer that interacts with an exterior surface of asubstrate and a monolayer that interacts with an interior surface of thesubstrate, where the monolayers join together at about the edge of theopening of the aperture and form a lipid bilayer spanning the substrateaperture (e.g., U.S. Pat. No. 7,777,505, entitled “Nanopore platformsfor ion channel recordings and single molecule detection and analysis,”naming White et al. as inventors).

In certain embodiments, a nanopore apparatus comprises a NanoporeMembrane System as described in U.S. patent application Ser. No.13/414,636 filed on Mar. 7, 2012, entitled “METHODS FOR VOLTAGE-INDUCEDPROTEIN INCORPORATION INTO PLANAR LIPID BILAYERS,” naming Ryan Dunnam,Geoffrey Barrall and Melissa Poquette as inventors, and designated byattorney docket no. EBS-1002-UT, the entirety of which herein isincorporated by reference, including all text, tables and drawings.

Conditions in which a polymer translocates through a protein pore (e.g.,a hemolysin polypeptide, a modified alpha hemolysin polypeptide, adevice comprising a hemolysin polypeptide) often include a suitableelectrolyte solution Any suitable electrolyte solution generally knowncan be used to translocate a polymer or to measure a CNR. Non-limitingexamples of electrolyte solutions include solutions comprising asuitable salt such as sodium chloride, potassium chloride, lithiumchloride, the like, or combinations thereof with concentrations rangingfrom 0.1 to 6 Molar (M) (e.g. 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 1 M, 2M, 3 M, 4 M, 5 M, or 6 M). In certain embodiments, a buffer is includedin an electrolyte solution to stabilize the pH. Any suitable buffer canbe used. In certain embodiments, a buffer comprises Tris at aconcentration of at least 300 mM, at least 200 mM, at least 100 mM, atleast 50 mM, at least 10 mM Tris or at least 1 mM. In some embodimentsan electrolyte solution comprises a suitable chelator such as EDTA at aconcentration of at least 0.1 mM, at least 0.5 mM or at least 1 mM. Incertain embodiments, a pH of an electrolyte solution ranges from 5 to 9(e.g. 5, 5.5, 6, 6.5, 7, 7.5, 8.0, 8.5, or 9.0). In certain embodiments,a pH of an electrolyte solution can range from about 7 to 7.5 (e.g. 7.0,7.1, 7.2, 7.3, 7.4, 7.5).

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1 Unmodified Alpha Hemolysin Amino Acid Sequence

The amino acid sequence for a wild type (WT) alpha hemolysinpolypeptide, which contains 293 amino acids as presented, is providedherein as SEQ ID NO: 1 for reference:

SEQ ID NO: 1 MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN SEQ ID NO: 2(YY-4S L135I D127K) MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDYKSYSSTLTYGFNGNVTGKDTGKIGGIIGANVSIGHSLSYYQPDFKTOLESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN SEQ ID NO: 3 (YY-4S L135I)MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDYKSYSSTLTYGFNGNVTGDDTGKIGGIIGANVSIGHSLSYYQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

Example 2

Below is a table of example polypeptides comprising a modified alphahemolysin amino acid sequence (Table 1). This list should in no way beconsidered limiting to the modifications that can be made to a referencealpha hemolysin reference amino acid sequence for the production of amodified alpha hemolysin amino acid sequence. The position numbers arein reference to SEQ ID NO: 1 provided in Example 1. The format is suchthat the term T109Y indicates that the native amino acid T109 (threonineat position 109) has been substituted with the non-native amino acid Y(tyrosine). In addition, L135I indicates that the beta barrel amino acidL135 (leucine at position 135) has been replaced with the differentamino acid I (isoleucine). The abbreviated names for some of themodified alpha hemolysin polypeptides are provided for reference to thedata presented in this application.

TABLE 1 Modified Alpha Hemolysin Protein Pore ABBREVIATED NAMET109Y/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S/V149Y YY-4SSDKMSI107Y/T109Y/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S/V149Y3Y-4S SDKMSI107Y/T109Y/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S/V149Y/P151Y4Y-4S SDKMS E111S/M113S/N121D/N123M/L135S/G147K/N139S/T145S/K147S/V149WV149W 4S SDKMST109Y/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S/V149W YW-4SSDKMS T109W/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S/V149YWY-4S SDKMST109W/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S/V149W WW-4SSDKMS I107W/T109W/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147SI107W T109W-4S SDKMST109W/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S T109W-4SSDKMS T109F/E111S/M113S/N121D/N123M/L135S/G137K/N139S/T145S/K147S/V149FFF-4S SDKMS T109Y/E111S/M113S/L135I/T145S/K147S/V149Y YY-4S L135IT109Y/E111S/M113S/D127K/L135I/T145S/K147S/V149Y YY-4S L135I D127KI107Y/T109Y/E111S/M113S/D127K/L135I/T145S/K147S/V149Y 3Y-4S L135I D127KT109Y/E111S/M113S/D127K/L135I/T145S/K147S Y109-4S L135I D127KE111S/M113S/D127K/L135I/T14S/K147S/V149Y Y149-4S L135I D127KT109Y/E111S/M113S/T125Q/D127K/L135I/T145S/K147S/V149Y YY-4S L135I T125QD127K T109Y/E111S/M113S/T125F/D127K/L135I/T145S/K147S/V149Y YY-4S T135IT125F D127K T109Y/E111S/M113S/T125S/D127K/L135I/T145S/K147S/V149Y YY-4SL135I T125S D127KT109Y/E111S/M113S/N121T/N123T/T125N/D127K/L135I/N139T/T145S/K147S/V149YYY-4S N3T L135I T125N D127KT109Y/E111D/M113S/N121S/D127K/L135I/N139S/V149Y YY-E111D M113S N2S L135ID127K T109Y/E111D/M113I/N121S/N123S/L135G/N139S/V149YT109Y/E111D/M113I/N121S/N123S/T125S/D127K/L135G/N139S/V149YT109Y/E111D/M113I/N121S/N123S/D127K/L135G/N139S/T145F/V149YT109Y/E111D/M113I/N121S/N123S/D127K/L135G/N139S/T145Q/V149YT109Y/E111D/M113I/N121S/N123S/D127K/L135G/N139S/T145S/V149YT109Y/E111S/M113S/N121D/N123M/T125S/L135S/G137K/N139S/T145S/K147S/V149YT109Y/E111S/M113S/N121D/N123M/T125S/L135F/G137K/N139S/T145S/K147S/V149YT109Y/E111S/M113S/N121D/N123M/T125S/L135O/G137K/N139S/T145S/K147S/V149YT109Y/E111S/M113S/N121S/N139S/T145S/K147S/V149Y YY-4S N25 T109Y/V149YYY-WT

Example 3

Provided in this Example 3 are descriptions of materials and proceduresutilized to generate results reported in Example 4 and Example 5.Example 4 and Example 5 describe results of experiments performed todemonstrate the translocation of a polymer through a modified alphahemolysin polypeptide and in some cases demonstrate slowing of thepolymer through the modified alpha hemolysin polypeptide compared to thealpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid. The slowing of the polymerthrough the modified alpha hemolysin polypeptide could be beneficial toreducing the measurement bandwidth used to record the polymertranslocating through the protein pore, thus improving the contrastsignal to noise ratio (CNR) of a protein pore, and enabling sequencingof polymers through the modified alpha hemolysin polypeptide.

Apparatus

Glass or quartz nanopore membranes (GNMs) were fabricated, the interiorof the GNM was filled with an electrolyte solution (e.g., 3 M NaCl(Sigma), 10 mM Tris, 2 mM EDTA and pH 7.2) and inserted horizontallythrough the wall of a polycarbonate cell into a fluid reservoir. AnAg/AgCl electrode produced by treating a 0.25 mm Ag wire with householdbleach was placed interior to the GNM. A holder provided a securemounting for the GNM, Ag/AgCl electrode interior to the GNM and provideda means of maintaining a constant back pressure on the GNM. The testcell had a reservoir of 250 μL and ports connected to syringes to allowfor raising and lowering the fluid level in the reservoir. A secondAg/AgCl electrode was placed in the test cell reservoir. The GNMelectrode and reference electrode were connected to a custom resistivefeedback headstage that allows for applying a voltage bias between theelectrodes and provides a low noise readout of the current between thetwo electrodes. All voltages were referenced with respect to theelectrode in the GNM. For example, a negative bias indicates that thetest cell reservoir electrode is at a negative potential with respect tothe electrode interior to the GNM. Data was acquired with a PCI-6251(National Instruments) DAQ card in a personal computer (Dell). A customLabView application handled voltage control, data acquisition, andsimple signal processing such as filtering.

Bilayer Formation

1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti) was dilutedin decane (Sigma) to a concentration of 5 mg/ml. A test cell reservoirwas filled to a level just above the face of the GNM with theelectrolyte solution described herein. A small drop (e.g., less thanabout 0.5μL) of the lipid/decane mixture was added to the surface ofelectrolyte. The fluid level in the test cell reservoir was then loweredbelow the face of the GNM and then raised above the face of the GNM.This action typically resulted in a bilayer although in some casesadditional lipid was added and the raising and lowering repeated.

Protein Pore Preparation

Modified alpha hemolysin protein monomers were generated through coupledin vitro transcription and translation (IVTT) using a bacterial extractkit (Promega) and then assembled into homo-heptamers on rabbit red bloodcell membranes (rRBCM) based on established protocols (B. Walker, and H.Bayley, “Key Residues for Membrane Binding, Oligomerization, and PoreForming Activity of Staphylococcal alpha hemolysin Identified byCysteine Scanning Mutagenesis and Targeted Chemical Modification,” J.Biol. Chem., vol. 270, no. 39, pp. 23065-23071, Sep. 29, 1995, 1995).Plasmid DNA (>95% supercoiled) of wild type and mutant alpha hemolysinwere made by GenScript. For most IVTT reactions, 4 micrograms of the DNA(Genscript) were mixed with contents of the kit according to themanufacturer's recommendation and supplemented with a mixture of acomplete set of amino acids and 4 microCi of S³⁵-Methionine (AmericanRadiolabeled Chemicals). The mixture was incubated at 37° C. for onehour, then mixed with rRBCM and further incubated for three hours. Atthe end of the incubation period, membranes were washed twice with MOPSbuffer followed by solubilization with SDS loading buffer. The latterwas loaded onto a 5% polyacrylamide gel and proteins separated byapplying a 60 V voltage overnight at room temperature. Gels were driedunder vacuum at 60° C. for 3-4 hours and exposed to X-ray film (Kodak)overnight at −80° C. Gels were developed manually using KodakDevelopment and Wash solutions. Bands corresponding to alpha hemolysinwere observed on the developed film due to the incorporation of theradioactive methionine. The film was used as a template to cut outportion of the dried gel containing the αHL protein. Proteins wererecovered from this portion by overnight electro-elution using anElutrap Electroelution system (GE Healthcare) and concentrated down to avolume of 10-20 microliters using microfuge concentrators (Millipore).Proteins were stored at −80° C. until use.

Alpha hemolysin incorporation in the bilayer was achieved by applying aback pressure (10-200 mmHg) to the interior of the GNM relative to thetest cell reservoir. The precise pressure applied was determined bymeasuring the pressure at which the bilayer fails and using a pressureabout 10 mmHg lower. After a single alpha hemolysin polypeptide wasincorporated as determined by a large jump in conducted current, thepressure was reduced to maintain a single protein insertion. Thisholding pressure was determined by measuring the pressure at which alphahemolysin was forced out of the bilayer. In some cases, the proteinconcentration was too low to allow for incorporation by applying a backpressure alone. In this case a high bias (>200 mV) was applied acrossthe bilayer to promote protein insertion, as described in a recentlyfiled U.S. patent application by EBS, U.S. Ser. No. 13/414,636.

Polymers

For the experiments presented, single stranded DNA was obtained fromGeneLink at 100 micromolar concentration in 10 mM Tris, 1 mM EDTA pH8.5. The strands obtained included polyC100 (SEQ ID NO: 4) and polyA100(SEQ ID NO: 5). After obtaining a protein insertion, the DNA was addedto the fluid reservoir of the test cell to allow the DNA to translocatethe alpha hemolysin polypeptides. Typically 1 to 20 microliters of DNAwas added to the test cell reservoir for a final DNA concentrationbetween 0.4 to 8.0 micromolar.

Example 4

The examples shown below demonstrate the increase in translocation timethat results from a modified alpha hemolysin polypeptide compared to theprotein pore without the at least one native amino acid substituted withthe non-native amino acid (e.g., a wild type alpha hemolysin protein).

Results

Below is a table showing the translocation times for the modified alphahemolysin polypeptide and the corresponding alpha hemolysin polypeptidewithout the at least one native amino acid substituted with thenon-native amino acid along with the polymer type (polyA100 (SEQ ID NO:5) or polyC100 (SEQ ID NO: 4)) and the percent increase in thetranslocation time between the two protein pores.

TABLE 2 Alpha Hemolysin Protein Pore without the at least One Native %Increase in Modified Alpha Hemolysin Translocation Time Amino AcidSubstituted with the Translocation Time Translocation Protein Pore(micro seconds) Non-Native Amino Acid (micro seconds) Time Polymer YY-4SSDKMS 362 4S SDKMS 102 354.9% polyC100 4Y-4S SDKMS 398 4S SDKMS 102390.2% polyC100 V149W 4S SDKMS 271 4S SDKMS 102 265.7% polyC100 YW-4SSDKMS 1135 4S SDKMS 102 1112.7% polyC100 WY-4S SDKMS 919 4S SDKMS 102901.0% polyC100 I107W T109W-4S SDKMS 249 4S SDKMS 102 244.1% polyC100YY-4S L135I D127K 420 4S L135I D127K 100 420.0% polyC100 YYY-4S L135ID127K 2100 4S L135I D127K 291 721.6% polyA100 YY-4S L135I T125Q D127K589 4S L135I T125Q D127K 278 211.9% polyA100 YY-4S L135I T125S D127K1170 4S L135I T125S D127K 156 750.0% polyA100 YY-4S L135I T125S D127K275 4S L135I T125S D127K 58 474.1% polyC100 YY-4S N3T L135I T125N D127K686 4S N3T L135I T125N D127K 163 420.9% polyA100 YY-E111D M113S N2SL135I D127K 400 4S N3T L135I T125N D127K 169 236.7% polyA100 YY-E111DM113S N2S L135I D127K 314 E111D M113S N2S L135I D127K 79 397.5% polyC100YY-WT 3941 WT 847 465.3% polyA100 YY-WT 1520 WT 214 710.3% polyC100

(TABLE 2 discloses “polyC100” and “polyA100” as SEQ ID NOS 4-5,respectively)

All data was obtained using a direct current (DC) measurement system.The data was collected with a 100 kHz bandwidth at −120 mV.

Example 5

The results below demonstrate that the modified alpha hemolysinpolypeptide can affect the computed CNR. These computations assume whitenoise, which is an accepted assumption for these measurements.

The CNR was computed in a manner consistent with the methods describedin patent applications PCT/US2012/04859 and PCT/US2012/04864. The CNRwas computed between homopolymers polyA100 (SEQ ID NO: 5) and polyC100(SEQ ID NO: 4). The DNA was added to the test cell and allowed totranslocate through the protein pore under a −120 mV DC bias and thedata was recorded with a 100 kHz low pass filter. The data was processedand filtered to the original predetermined filter frequency of 10 kHz.The contrast was computed as the difference between the average polyA100(SEQ ID NO: 5) level and the average polyC100 (SEQ ID NO: 4) level. Thenoise was then computed as the average RMS noise for polyA100 (SEQ IDNO: 5) at 10 kHz and the average RMS noise for polyC100 (SEQ ID NO: 4)at 10 kHz. A total noise value was computed by taking the square root ofthe sum of the squares of the RMS noise values for polyA100 (SEQ ID NO:5) and polyC100 (SEQ ID NO: 4). In addition, a tmax (average duration)value was obtained for polyA100 (SEQ ID NO: 5) and separately polyC100(SEQ ID NO: 4) events. The CNR was then computed by dividing thecontrast by the total noise values. The CNR values for the modifiedalpha hemolysin polypeptide compared to the protein pore without the atleast one native amino acid substituted with the non-native amino acidare shown in Table 3.

TABLE 3 Alpha Hemolysin Protein Tmax Pore without the at least TmaxModified Alpha Total polyA100 One Native Amino Acid Total polyA100Hemolysin Noise (micro Substituted with the Non- Noise (micro ProteinPore CNR (10 kHz) CNR seconds) Native Amino Acid Contrast (10 kHz) CNRseconds) YY-4S L135I 13.3 4.75 2.8 1458 4S L135I D127K 10.4 2.08 5.0 279D127K YY-4S L135I 7.5 5.88 1.3 1156.0 4S L135I T125S D127K 8.6 3.01 2.9154 T125S D127K

(TABLE 3 discloses “polyA100” as SEQ ID NO: 5)

The data show that in some cases, the CNR at the same measurementsbandwidth between the modified alpha hemolysin polypeptide and theprotein pore without the at least one native amino acid substituted withthe non-native amino acid decreases. However, the increase intranslocation time enables a lower measurement bandwidth (predeterminedfilter frequency) to be utilized, which will result in lower totalnoise. Thus, a calculation assuming white noise is done to determine theoverall effect on the CNR as a result of this reduced total noise. Thecalculation is as follows:Weighted CNR=CNR*sqrt(tmax)

Table 4 below shows the Weighted CNRs taking into account thetranslocation time, tmax. In addition, a ratio between the Weighted CNRof the modified alpha hemolysin polypeptide to the protein pore withoutthe at least one native amino acid substituted with the non-native aminoacid is shown. If this ratio is greater than 1, it indicates that theCNR of the modified protein pore is improved as a result of the increasein translocation time. This is due to the fact that the reduction inbandwidth allowed will decrease the total noise sufficiently to resultin an overall increase in CNR. This overall increase in CNR can helpenable polymer sequencing and improve sequencing accuracy.

TABLE 4 Alpha Hemolysin Protein Ratio of Tmax Pore without the at leastTmax Modified Modified Alpha polyA100 One Native Amino Acid polyA100Pore CNR to Hemolysin (milli Weighted Substituted with the Non- (milliWeighted Unmodifed Protein Pore CNR seconds) CNR Native Amino AcidContrast seconds) CNR Pore CNR YY-4S L135I 2.8 1.458 3.38 4S L135I D127K5.0 0.279 2.64 1.28 D127K YY-4S L135I 1.3 1.2 1.40 4S L135I T125S D127K2.9 0.154 1.14 1.23 T125S D127K

(TABLE 4 discloses “polyA100” as SEQ ID NO: 5)

Example 6 Examples of Embodiments

Provided hereafter are non-limiting examples of certain embodiments ofthe technology.

A1. A polypeptide comprising a modified alpha hemolysin amino acidsequence, wherein:

-   -   the modified alpha hemolysin amino acid sequence comprises one        or more native amino acid substitutions at one or more positions        corresponding to positions 1-109 and 149-293 of SEQ ID NO: 1;        and    -   at least one of the one or more native amino acid substitutions        independently is to a non-native hydrophobic amino acid or        non-native aromatic amino acid, or non-native aromatic and        hydrophobic amino acid.

A2. A polypeptide comprising a modified alpha hemolysin amino acidsequence, wherein:

-   -   the modified alpha hemolysin amino acid sequence comprises one        or more native amino acid substitutions at one or more positions        corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 97, 99, 101, 103, 105, 107, 109, 149, 151,        153, 155, 157, 159, 161, 163, 165, 167, 169, 225, 227, 229, 231        or 233 of SEQ ID NO: 1; and    -   at least one of the one or more native amino acid substitutions        independently is to a non-native hydrophobic amino acid or        non-native aromatic amino acid, or non-native aromatic and        hydrophobic amino acid.

A3. A polypeptide comprising a modified alpha hemolysin amino acidsequence, wherein:

-   -   the modified alpha hemolysin amino acid sequence comprises one        or more native amino acid substitutions at one or more positions        corresponding to positions 105, 107, 109, 149, 151 or 153 of SEQ        ID NO: 1; and    -   at least one of the one or more native amino acid substitutions        independently is to a non-native hydrophobic amino acid or        non-native aromatic amino acid, or non-native aromatic and        hydrophobic amino acid.

A4. A polypeptide comprising a modified alpha hemolysin amino acidsequence, wherein:

-   -   the modified alpha hemolysin amino acid sequence comprises one        or more native amino acid substitutions at one or more positions        corresponding to positions 107, 109, 149 or 151 of SEQ ID NO: 1;        and    -   at least one of the one or more native amino acid substitutions        independently is to a non-native hydrophobic amino acid or        non-native aromatic amino acid, or non-native aromatic and        hydrophobic amino acid.

A5. A polypeptide comprising a modified alpha hemolysin amino acidsequence, wherein:

-   -   the modified alpha hemolysin amino acid sequence comprises one        or more native amino acid substitutions at one or more positions        corresponding to positions 109 or 149 of SEQ ID NO: 1; and    -   at least one of the one or more native amino acid substitutions        independently is to a non-native hydrophobic amino acid or        non-native aromatic amino acid, or non-native aromatic and        hydrophobic amino acid.

A5a. The polypeptide of any one of embodiments A1 to A5, wherein themodified alpha hemolysin amino acid sequence is modified relative to anamino acid sequence of a reference alpha hemolysin polypeptide.

A5a.1 . The polypeptide of any one of embodiments A1 to A5, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 90% identity to SEQ ID NO: 1.

A5a.2. The polypeptide of any one of embodiments A1 to A5, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 95% identity to SEQ ID NO: 1.

A5a.3. The polypeptide of any one of embodiments A1 to A5, wherein theamino acid sequence of the reference alpha hemolysin polypeptideconsists of the amino acid sequence of SEQ ID NO: 1.

A5a.4. The polypeptide of any one of embodiments A1 to A5, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 90% homology to SEQ ID NO: 1.

A5a.5. The polypeptide of any one of embodiments A1 to A5, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 95% homology to SEQ ID NO: 1.

A5.1. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5a wherein the non-nativeamino acid is hydrophobic.

A5.2. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5a wherein the non-nativeamino acid is aromatic.

A5.3. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5.2 wherein the non-nativeamino acid is hydrophobic and aromatic.

A5.4. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5.3 wherein the non-nativeamino acid is larger than the native amino acid.

A5.5. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5.4, wherein the non-nativeamino acid is naturally occurring.

A5.6. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5.4, wherein the non-nativeamino acid is synthetic.

A6. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5.1, 5.4 or 5.5, wherein thenon-native amino acid is methionine (M).

A7. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A5.1, A5.3, 5.4 and 5.5,wherein the non-native amino acid is selected from the group consistingof phenylalanine (F), tryptophan (W) and tyrosine (Y).

A8. The polypeptide comprising a modified alpha hemolysin amino acidsequence of embodiment A7, wherein the non-native amino acid isphenylalanine (F).

A9. The polypeptide comprising a modified alpha hemolysin amino acidsequence of embodiment A7, wherein the non-native amino acid istryptophan (W).

A10. The polypeptide comprising a modified alpha hemolysin amino acidsequence of embodiment A7, wherein the non-native amino acid is tyrosine(Y).

A11. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment wherein at least two native aminoacids are substituted with non-native amino acids.

A12. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein at least three nativeamino acids are substituted with non-native amino acids.

A13. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein at least four native aminoacids are substituted with non-native amino acids.

A13.1. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A11 to A13, wherein the non-nativeamino acids are the same.

A13.2. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A11 to A13, wherein the non-nativeamino acids are not the same.

A13.3. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A12 to A13, wherein at least two ofthe non-native amino acids are the same.

A14. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein at least one beta barrelamino acid at any beta barrel amino acid position 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 or147 is replaced with a different amino acid.

A15. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein at least two beta barrelamino acids at any beta barrel amino acid position 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 or147 are replaced with a different amino acid.

A16. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein at least three beta barrelamino acids at any beta barrel amino acid position 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 or147 are replaced with a different amino acid.

A17. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein at least four beta barrelamino acids at any beta barrel amino acid position 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 or147 are replaced with a different amino acid.

A18. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein at least five beta barrelamino acids at any beta barrel amino acid position 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 or147 are replaced with a different amino acid.

A18.1. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A14 to A18, wherein the differentamino acid is a naturally occurring amino acid.

A18.2. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A14 to A18, wherein the differentamino acid is a synthetic amino acid.

A19. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least 20percent compared to the translocation time of the polymer through analpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid.

A20. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least 50percent compared to the translocation time of the polymer through analpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid.

A21. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least100 percent compared to the translocation time of the polymer through analpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid.

A22. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least200 percent compared to the translocation time of the polymer through analpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid.

A23. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least300 percent compared to the translocation time of the polymer through analpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid.

A24. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least400 percent compared to the translocation time of the polymer through analpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid.

A24. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least500 percent compared to the translocation time of the polymer through analpha hemolysin polypeptide without the at least one native amino acidsubstituted with the non-native amino acid.

A25. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the at least one nativeamino acid substituted with the non-native amino acid enables anincrease in a translocation time of a polymer through the polypeptidecomprising a modified alpha hemolysin amino acid sequence by at least1000 percent compared to the translocation time of the polymer throughan alpha hemolysin polypeptide without the at least one native aminoacid substituted with the non-native amino acid.

A26. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A19 to A25 that permits a measurementof a first level and a second level within a residual current of thepolypeptide comprising a modified alpha hemolysin amino acid sequence,as the polymer translocates through the polypeptide comprising amodified alpha hemolysin amino acid sequence, with a contrast signal tonoise ratio (CNR) computed at a predetermined filter frequency;

-   -   which polymer comprises two or more sections, each of which        sections comprises at least a portion of a monomer;    -   which CNR is calculated as a contrast signal divided by a noise        value;    -   which contrast signal is calculated as the difference between        the first level and the second level, wherein:    -   each level used for calculating the CNR correlates to a        composition of a section of the polymer,    -   the first level and the second level are measurably distinct;        and    -   which noise value is computed at the predetermined filter        frequency.

A27. The polypeptide comprising a modified alpha hemolysin amino acidsequence of embodiment A26, wherein the increase in the translocationtime enables a lower predetermined filter frequency to be used in thecomputation of the CNR.

A28. The polypeptide comprising a modified alpha hemolysin amino acidsequence of embodiment A27, wherein the lower predetermined filterfrequency results in a lower noise value.

A29. The polypeptide comprising a modified alpha hemolysin amino acidsequence of embodiment A28, wherein the lower noise value results in ahigher CNR.

A29.1 The polypeptide comprising a modified alpha hemolysin amino acidsequence of embodiment A27, wherein the lower predetermined filterfrequency results in a higher Weighted CNR.

A30. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the native amino acid T109is substituted with the non-native amino acid tyrosine (T109Y).

A31. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the native amino acid V149is substituted with the non-native amino acid tyrosine (V149Y).

A32. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any preceding embodiment, wherein the native amino acidsT109 and V149 are substituted with the non-native amino acid tyrosine(T109Y and V149Y).

A33. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A1 to A32 wherein the native aminoacids T109 and V149 are substituted with the non-native amino acidstyrosine and tryptophan respectively (T109Y and V149W).

A34. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A14 to A33, wherein the native aminoacids T109 and V149 are substituted with the non-native amino acidtyrosine (T109Y and V149Y) and wherein the beta barrel amino acids E111,M113, D127, L135, T145 and K147 are substituted with the different aminoacids serine, serine, lysine, isoleucine, serine and serine respectively(E111 S, M113S, D127K, L135I, T145S, and K147S).

A35. The polypeptide comprising a modified alpha hemolysin amino acidsequence of any one of embodiments A14 to A33, wherein the native aminoacids T109 and V149 are substituted with the non-native amino acidtyrosine (T109Y and V149Y) and wherein the beta barrel amino acids E111,M113, L135, T145 and K147 are substituted with the different amino acidsserine, serine, isoleucine, serine and serine respectively (E111S,M113S, L135I, T145S, and K147S).

A36. The polypeptide of any preceding embodiment, wherein the modifiedalpha hemolysin sequence comprises the SEQ ID NO: 1 except for the oneor more native amino acids substitutions and the one or more differentamino acid replacements.

A37. The polypeptide of any one of embodiments A1 to A36, wherein thepolypeptide is an isolated polypeptide.

A38. The polypeptide of embodiment A1, comprising the modified alphahemolysin amino acid sequence of SEQ ID NO: 2.

A39. The polypeptide of embodiment A1, comprising the modified alphahemolysin amino acid sequence of SEQ ID NO: 3.

A40. The polypeptide of any one of embodiments A1 to A37, comprising anamino acid sequence with 90% identity to SEQ ID NO: 2.

A41. The polypeptide of any one of embodiments A1 to A37, comprising anamino acid sequence with 90% identity to SEQ ID NO: 3.

B1. The method of using the polypeptide comprising a modified alphahemolysin amino acid sequence of any one of embodiments A1 to A39 tosequence a polymer.

C1. The method of increasing the translocation time of a polymer using apolypeptide comprising a modified alpha hemolysin amino acid sequence ofany one of embodiments A1 to A40, relative to a translocation time ofthe polymer through a reference alpha hemolysin polypeptide.

D1. The method of translocating a polymer through a polypeptidecomprising a modified alpha hemolysin amino acid sequence of any one ofembodiments A1 to A37.

E1. A modified alpha hemolysin polypeptide comprising a modified alphahemolysin amino acid sequence, wherein:

-   -   the modified alpha hemolysin amino acid sequence comprises one        or more amino acid substitutions at one or more positions        corresponding to positions 1-109 and 149-293 of SEQ ID NO: 1;        and    -   at least one of the one or more amino acid substitutions        independently is to a non-native amino acid, wherein the        non-native amino acid is hydrophobic or aromatic, or hydrophobic        and aromatic.

E2. The polypeptide of embodiment E1, wherein the modified alphahemolysin amino acid sequence comprises one or more substitutionslocated within a beta barrel.

E3. The polypeptide of embodiment E2, wherein the beta barrel comprisespositions 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145 and 147 of SEQ ID NO: 1.

E4. The polypeptide of embodiment E2 or E3, wherein the modified alphahemolysin amino acid sequence comprises at least two amino acidsubstitutions located in the beta barrel.

E5. The polypeptide of any one of embodiments E2 to E4, wherein themodified alpha hemolysin amino acid sequence comprises at least threeamino acid substitutions located in the beta barrel.

E6. The polypeptide of any one of embodiments E2 to E5, wherein themodified alpha hemolysin amino acid sequence comprises at least fouramino acid substitutions located in the beta barrel.

E7. The polypeptide of any one of embodiments E2 to E6, wherein themodified alpha hemolysin amino acid sequence comprises at least fiveamino acid substitutions located in the beta barrel.

E8. The polypeptide of embodiment E1, wherein the one or more positionscorrespond to positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 97, 99, 101, 103, 105, 107, 109, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 225, 227, 229, 231 or 233 of SEQ ID NO: 1.

E9. The polypeptide of embodiment E1, wherein the one or more positionscorrespond to positions 105, 107, 109, 149, 151 or 153 of SEQ ID NO: 1.

E10. The polypeptide of embodiment E1, wherein the one or more positionscorrespond to positions 107, 109, 149 or 151 of SEQ ID NO: 1.

E11 . The polypeptide of embodiment E1, wherein the one or morepositions correspond to positions 109 or 149 of SEQ ID NO: 1.

E12. The polypeptide of any one of embodiments E1 to E11, wherein thenon-native amino acid is hydrophobic.

E13. The polypeptide of any one of embodiments E1 to E12, wherein thenon-native amino acid is aromatic.

E14. The polypeptide of any one of embodiments E1 to E13, wherein thenon-native amino acid is hydrophobic and aromatic.

E15. The polypeptide of any one of embodiments E1 to E14, wherein theone or more amino acid substitutions independently is to one or morelarger amino acids.

E16. The polypeptide of any one of embodiments E1 to E15, wherein theone or more amino acid substitutions independently is to one or morenaturally occurring amino acids.

E17. The polypeptide of any one of embodiments E1 to E16, wherein theone or more amino acid substitutions independently is to one or moresynthetic amino acids.

E18. The polypeptide of any one of embodiments E1 to E7, E10 and E11,wherein the one or more amino acid substitutions independently is to oneor more methionines.

E19. The polypeptide of any one of embodiments E1 to E13, E15 and E16,wherein the non-native amino acid is selected from the group consistingof phenylalanine (F), tryptophan (W) and tyrosine (Y).

E20. The polypeptide of embodiment E19, wherein the non-native aminoacid is phenylalanine (F).

E21. The polypeptide of embodiment E19, wherein the non-native aminoacid is tryptophan (W).

E22. The polypeptide of embodiment E19, wherein the non-native aminoacid is tyrosine (Y).

E23. The polypeptide of any one of embodiments E1 to E22, wherein themodified alpha hemolysin amino acid sequence comprises at least twoamino acid substitutions.

E24. The polypeptide of any one of embodiments E1 to E22, wherein themodified alpha hemolysin amino acid sequence comprises at least threeamino acid substitutions

E25. The polypeptide of any one of embodiments E1 to E22, wherein themodified alpha hemolysin amino acid sequence comprises at least fouramino acid substitutions.

E26. The polypeptide of embodiment E23, wherein the at least two aminoacid substitutions are to non-native amino acids that are the same.

E27. The polypeptide of embodiment E23, wherein the at least two aminoacid substitutions are to non-native amino acids that are not the same.

E28. The polypeptide of embodiment E23, wherein the at least two aminoacid substitutions are to at least two non-native amino acids that arenot the same.

E29. The polypeptide of any one of embodiments E1 to E28, comprising afirst translocation time determined for a polymer translocating throughthe polypeptide, wherein the first translocation time is at least 20%longer than a second translocation time for the polymer as determinedfor a reference alpha hemolysin protein.

E29.1. The polypeptide of any one of embodiments E1 to E28, comprising afirst translocation time determined for a polymer translocating throughthe polypeptide, wherein the first translocation time is at least 20%longer than a second translocation time for the polymer as determinedfor a reference alpha hemolysin protein comprising one or more aminoacid substitutions located in a beta barrel.

E30. The polypeptide of embodiment E29 or E29.1, wherein the firsttranslocation time is at least 50% longer than the second translocationtime.

E31. The polypeptide of embodiment E29 or E29.1, wherein the firsttranslocation time is at least 100% longer than the second translocationtime.

E32. The polypeptide of embodiment E29 or E29.1, wherein the firsttranslocation time is at least 200% longer than the second translocationtime.

E33. The polypeptide of embodiment E29 or E29.1, wherein the firsttranslocation time is at least 300% longer than the second translocationtime.

E34. The polypeptide of embodiment E29 or E29.1, wherein the firsttranslocation time is at least 400% longer than the second translocationtime.

E35. The polypeptide of embodiment E29 or E29.1, wherein the firsttranslocation time is at least 500% longer than the second translocationtime.

E36. The polypeptide of embodiment E29 or E29.1, wherein the firsttranslocation time is at least 1000% longer than the secondtranslocation time.

E37. The polypeptide of any one of embodiments E29 to E36, wherein thepolymer is a polynucleotide.

E38. The polypeptide of embodiment E37, wherein the polynucleotideconsists of polyA, polyC, polyT, polyU or polyG.

E39. The polypeptide of any one of embodiments E29 to E36, wherein thepolymer is a polypeptide.

E40. The polypeptide of any one of embodiments E29 to E39, wherein thereference alpha hemolysin protein comprises the sequence of SEQ ID NO:1.

E41. The polypeptide of any one of embodiments E29 to E40 that permits ameasurement of a first level and a second level within a residualcurrent of the polypeptide, as the polymer translocates through thepolypeptide with a contrast signal to noise ratio (CNR) computed at apredetermined filter frequency;

-   -   which polymer comprises two or more sections, each of which        sections comprises at least a portion of a monomer;    -   which CNR is calculated as a contrast signal divided by a noise        value;    -   which contrast signal is calculated as the difference between        the first level and the second level, wherein:    -   each level used for calculating the CNR correlates to a        composition of a section of the polymer,    -   the first level and the second level are measurably distinct;        and    -   which noise value is computed at the predetermined filter        frequency.

E42. The polypeptide of embodiment E41, wherein the increase in thetranslocation time enables a lower predetermined filter frequency to beused in the computation of the CNR.

E43. The polypeptide of embodiment E42, wherein the lower predeterminedfilter frequency results in a lower noise value.

E44. The polypeptide of embodiment E43, wherein the lower noise valueresults in a higher CNR.

E45. The polypeptide of any one of embodiments E1 to E44, whereinthreonine at position 109 is substituted with tyrosine.

E46. The polypeptide of any one of embodiments E1 to E46, wherein valineat position 149 is substituted with tyrosine.

E47. The polypeptide of any one of embodiments E1 to E46, whereinthreonine at position 109 is substituted with tyrosine and valine atposition 149 is substituted with tyrosine.

E48. The polypeptide of any one of embodiments E1 to E45, whereinthreonine at position 109 is substituted with tyrosine and valine atposition 149 is substituted with tryptophan.

E49. The polypeptide of embodiment E47, wherein glutamate, methionine,aspartic acid, leucine, threonine and lysine at positions 111, 113, 127,135, 145 and 147, respectively, are substituted with serine, serine,lysine, isoleucine, serine and serine respectively.

E50. The polypeptide of embodiment E47, wherein glutamate, methionine,leucine, threonine and lysine at positions 111, 113, 135, 145 and 147,respectively, are substituted with serine, serine, isoleucine, serineand serine respectively.

E51. The polypeptide of any one of embodiments E1 to E50, wherein themodified alpha hemolysin sequence comprises the sequence of SEQ ID NO: 1except for the one or more amino acid substitutions.

E52. The polypeptide of any one of embodiments E1 to E51, wherein thepolypeptide is an isolated polypeptide.

E53. The polypeptide of any one of embodiments E1 to E52, wherein themodified alpha hemolysin amino acid sequence is modified relative to anamino acid sequence of a reference alpha hemolysin polypeptide.

E54. The polypeptide of any one of embodiments E1 to E52, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 90% identity to SEQ ID NO: 1.

E55. The polypeptide of any one of embodiments E1 to E52, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 95% identity to SEQ ID NO: 1.

E56. The polypeptide of any one of embodiments E1 to E52, wherein theamino acid sequence of the reference alpha hemolysin polypeptideconsists of the amino acid sequence of SEQ ID NO: 1.

E57. The polypeptide of any one of embodiments E1 to E52, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 90% homology to SEQ ID NO: 1.

E58. The polypeptide of any one of embodiments E1 to E52, wherein theamino acid sequence of the reference alpha hemolysin polypeptidecomprises at least 95% homology to SEQ ID NO: 1.

E59. The polypeptide of embodiment E1, comprising the modified alphahemolysin amino acid sequence of SEQ ID NO: 2.

E60. The polypeptide of embodiment E1, comprising the modified alphahemolysin amino acid sequence of SEQ ID NO: 3.

E61. The polypeptide of any one of embodiments E1 to E58, comprising anamino acid sequence with 90% identity to SEQ ID NO: 2.

E62. The polypeptide of any one of embodiments E1 to E58, comprising anamino acid sequence with 90% identity to SEQ ID NO: 3.

F1. A method of sequencing a polymer with a modified alpha hemolysinpolypeptide comprising:

-   -   (a) contacting a polymer with a modified alpha hemolysin        polypeptide, wherein the modified alpha hemolysin polypeptide        comprises one or more amino acid substitutions relative to an        amino acid sequence of a reference alpha hemolysin protein, and    -   (b) determining the sequence of the polymer according to one or        more electrical changes across or through the modified alpha        hemolysis protein pore.

F2. The method of embodiment F1, wherein the reference alpha hemolysisprotein comprises the sequence of SEQ ID NO:1.

F3. The method of embodiment F1 or F2, wherein the modified alphahemolysin polypeptide comprises one or more amino acid substitutionsselected from one or more positions corresponding to amino acids 1-109and 149-293 of SEQ ID NO: 1, wherein the one or more amino acids aresubstituted with a non-native hydrophobic amino acid, non-nativearomatic amino acid, or non-native aromatic and hydrophobic amino acid.

F4. The method of embodiment F3, wherein the one or more amino acidsubstitutions are selected from one or more positions of 1 to 16, 97,99, 101, 103, 105, 107, 109, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 225, 227, 229, 231 or 233 of SEQ ID NO: 1.

F5. The method of embodiment F3, wherein one or more amino acidssubstitutions are selected from one or more positions of 105, 107, 109,149, 151 or 153 of SEQ ID NO: 1.

F6. The method of embodiment F3, wherein one or more amino acidssubstitutions are selected from one or more positions of 107, 109, 149or 151 of SEQ ID NO: 1.

F7. The method of embodiment F2, wherein one or more amino acidssubstitutions are selected from one or more positions of 109 or 149 ofSEQ ID NO: 1 .

F8. The method of any one of embodiments F1 to F7, wherein the referencealpha hemolysin protein comprises one or more amino acid substitutionslocated in a beta barrel.

F9. The method of any one of embodiments F1 to F8, wherein the modifiedalpha hemolysin polypeptide is a modified alpha hemolysin polypeptide ofany one of embodiments E1 to E61.

G1. A method for translocating a polymer through a modified alphahemolysin polypeptide comprising: contacting a polymer with a modifiedalpha hemolysin polypeptide under conditions in which the polymertranslocates through the modified alpha hemolysin polypeptide,

-   -   wherein the modified alpha hemolysin polypeptide comprises an        amino acid sequence of a reference alpha hemolysin protein        comprising one or more amino acid substitutions, and    -   determining a translocation time of the polymer through the        modified alpha hemolysin polypeptide.

G1.2. The method of embodiment G1, wherein the translocation time of thepolymer through the modified alpha hemolysin polypeptide is at least 20%longer than a translocation time of the polymer through the referencealpha hemolysin protein.

G2. The method of embodiment G1 or G1.2, wherein the reference alphahemolysis protein comprises the sequence of SEQ ID NO:1.

G3. The method of embodiment G2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 1-109 and 149-293 of SEQ ID NO: 1 are substituted with anon-native hydrophobic amino acid, non-native aromatic amino acid, ornon-native aromatic and hydrophobic amino acid.

G4. The method of embodiment G2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 1 to 16, 97, 99, 101, 103, 105, 107, 109, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 225, 227, 229, 231 or 233 of

SEQ ID NO: 1 are substituted with a non-native hydrophobic amino acid,non-native aromatic amino acid, or non-native aromatic and hydrophobicamino acid.

G5. The method of embodiment G2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 105, 107, 109, 149, 151 or 153 of SEQ ID NO: 1 aresubstituted with a non-native hydrophobic amino acid or non-nativearomatic amino acid, or non-native aromatic and hydrophobic amino acid.

G6. The method of embodiment G2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 107, 109, 149 or 151 of SEQ ID NO: 1 are substituted with anon-native hydrophobic amino acid or non-native aromatic amino acid, ornon-native aromatic and hydrophobic amino acid.

G7. The method of embodiment G2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 109 or 149 of SEQ ID NO: 1 are substituted with anon-native hydrophobic amino acid, non-native aromatic amino acid, ornon-native aromatic and hydrophobic amino acid.

G8. The method of any one of embodiments G1 to G7, wherein the referencealpha hemolysin protein comprises one or more amino acid substitutionslocated in a beta barrel.

G9. The method of any one of embodiments G1 to G8, wherein the modifiedalpha hemolysin polypeptide is a modified alpha hemolysin polypeptide ofany one of embodiments E1 to E52.

H1. A method of increasing the translocation time of a polymer through amodified alpha hemolysin polypeptide comprising:

-   -   (a) substituting one or more amino acids of a reference alpha        hemolysin polypeptide, wherein a modified alpha hemolysin        polypeptide is generated, and    -   (b) contacting the modified alpha hemolysin polypeptide with a        polymer under conditions in which the polymer translocates        through the modified alpha hemolysin polypeptide.

H1.1. The method of embodiment H1, wherein the translocation time of thepolymer through the modified alpha hemolysin polypeptide is at least 20%longer than a translocation time of the polymer through the referencealpha hemolysin protein.

H2. The method of embodiment H1 or H1.1, wherein the reference alphahemolysis protein comprises the sequence of SEQ ID NO:1.

H3. The method of embodiment H2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 1-109 and 149-293 of SEQ ID NO: 1 are substituted with anon-native hydrophobic amino acid, non-native aromatic amino acid, ornon-native aromatic and hydrophobic amino acid.

H4. The method of embodiment H2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 1 to 16, 97, 99, 101, 103, 105, 107, 109, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 225, 227, 229, 231 or 233 of SEQID NO: 1 are substituted with a non-native hydrophobic amino acid,non-native aromatic amino acid, or non-native aromatic and hydrophobicamino acid.

H5. The method of embodiment H2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 105, 107, 109, 149, 151 or 153 of SEQ ID NO: 1 aresubstituted with a non-native hydrophobic amino acid or non-nativearomatic amino acid, or non-native aromatic and hydrophobic amino acid.

H6. The method of embodiment H2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 107, 109, 149 or 151 of SEQ ID NO: 1 are substituted with anon-native hydrophobic amino acid or non-native aromatic amino acid, ornon-native aromatic and hydrophobic amino acid.

H7. The method of embodiment H2, wherein, for the modified alphahemolysin polypeptide, one or more amino acids selected from one or morepositions of 109 or 149 of SEQ ID NO: 1 are substituted with anon-native hydrophobic amino acid, non-native aromatic amino acid, ornon-native aromatic and hydrophobic amino acid.

H8. The method of any one of embodiments H1 to H7, wherein the referencealpha hemolysin protein comprises one or more amino acid substitutionslocated in a beta barrel.

H9. The method of any one of embodiments H1 to H8, wherein the modifiedalpha hemolysin polypeptide is a modified alpha hemolysin polypeptide ofany one of embodiments E1 to E52.

I1. A nanopore device comprising a modified alpha hemolysin protein ofany one of embodiments E1 to E52.

I2. The nanopore device of embodiment I1, wherein the modified alphahemolysin protein comprises an amino acid sequence of a reference alphahemolysis protein with one or more amino acid substitutions.

I3. The nanopore device of embodiment I2, wherein the reference alphahemolysin protein comprises an amino acid sequence of SEQ ID NO: 1.

I4. The nanopore device of embodiment I3, wherein, for the modifiedalpha hemolysin polypeptide, one or more amino acids selected from oneor more positions of 1-109 and 149-293 of SEQ ID NO: 1 are substitutedwith a non-native hydrophobic amino acid, non-native aromatic aminoacid, or non-native aromatic and hydrophobic amino acid.

I5. The nanopore device of embodiment I3, wherein, for the modifiedalpha hemolysin polypeptide, one or more amino acids selected from oneor more positions of 1 to 16, 97, 99, 101, 103, 105, 107, 109, 149, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 225, 227, 229, 231 or 233of SEQ ID NO: 1 are substituted with a non-native hydrophobic aminoacid, non-native aromatic amino acid, or non-native aromatic andhydrophobic amino acid.

I6. The nanopore device of embodiment I3, wherein, for the modifiedalpha hemolysin polypeptide, one or more amino acids selected from oneor more positions of 105, 107, 109, 149, 151 or 153 of SEQ ID NO: 1 aresubstituted with a non-native hydrophobic amino acid or non-nativearomatic amino acid, or non-native aromatic and hydrophobic amino acid.

I7. The nanopore device of embodiment I3, wherein, for the modifiedalpha hemolysin polypeptide, one or more amino acids selected from oneor more positions of 107, 109, 149 or 151 of SEQ ID NO: 1 aresubstituted with a non-native hydrophobic amino acid or non-nativearomatic amino acid, or non-native aromatic and hydrophobic amino acid.

I8. The nanopore device of embodiment I3, wherein, for the modifiedalpha hemolysin polypeptide, one or more amino acids selected from oneor more positions of 109 or 149 of SEQ ID NO: 1 are substituted with anon-native hydrophobic amino acid, non-native aromatic amino acid, ornon-native aromatic and hydrophobic amino acid.

I9. The nanopore device of any one of embodiments I1 to I8, wherein thereference alpha hemolysin protein comprises one or more amino acidsubstitutions located in a beta barrel.

I10. The nanopore device of any one of embodiments I1 to I9, wherein themodified alpha hemolysin polypeptide is a modified alpha hemolysinpolypeptide of any one of embodiments E1 to E52.

I11. The nanopore device of any one of embodiments I1 to I10, comprisinga lipid bilayer in which the modified alpha hemolysin polypeptideresides.

* * *

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed is:
 1. A modified Staphylococcus aureus alpha hemolysinpolypeptide comprising: a beta barrel region; and one or more amino acidsubstitutions of a non-native amino acid for a native amino acid at oneor more positions corresponding to positions 107, 109, 151 or 153 of awild-type Staphylococcus aureus alpha hemolysin polypeptide, wherein theamino acid residues of the wild-type Staphylococcus aureus alphahemolysin polypeptide align with positions of the amino acid sequence ofthe Staphylococcus aureus alpha hemolysin polypeptide of SEQ ID NO. 1,the non-native amino acids are hydrophobic or aromatic, or hydrophobicand aromatic, and the substitutions result in a modified hemolysinpolypeptide, whereby translocation of a polymer through the modifiedpolypeptide is slower than translocation through the polypeptide withoutthe one or more amino acid substitutions .
 2. The polypeptide of claim1, wherein the non-native amino acids are hydrophobic.
 3. Thepolypeptide of claim 1, wherein the non-native amino acids are aromatic.4. The polypeptide of claim 1, wherein the non-native amino acids arehydrophobic and aromatic.
 5. The polypeptide of claim 1, wherein atleast one of the non-native amino acids is tryptophan (W).
 6. Thepolypeptide of claim 1, wherein at least one of the non-native aminoacids is tyrosine (Y).
 7. The polypeptide of claim 1, comprising one ormore substitutions at positions that are part of the sequence of thebeta barrel region, wherein the beta barrel region comprises positions111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145 and 147 of a wild-type Staphylococcus aureus alphahemolysin polypeptide and the amino acid residues of the wild-typeStaphylococcus aureus alpha hemolysin polypeptide align with positionsof the amino acid sequence of the Staphylococcus aureus alpha hemolysinpolypeptide of SEQ ID NO.
 1. 8. The polypeptide of claim 7, wherein theone or more substitutions at positions that are part of the sequence ofthe beta barrel region comprises position
 135. 9. The polypeptide ofclaim 1, further comprising an amino acid substitution of a non-nativeamino acid for a native amino acid at a position corresponding toposition 149 of a wild-type Staphylococcus aureus alpha hemolysinpolypeptide, wherein the amino acid residues of the wild-typeStaphylococcus aureus alpha hemolysin polypeptide align with positionsof the amino acid sequence of the Staphylococcus aureus alpha hemolysinpolypeptide of SEQ ID NO. 1, the non-native amino acid is hydrophobic oraromatic and the substitution results in a modified hemolysinpolypeptide, whereby translocation of a polymer through the modifiedpolypeptide is slower than translocation through the polypeptide withoutthe amino acid substitution.
 10. The polypeptide of claim 9, wherein thepositions correspond to positions 109 and 149 of a wild-typeStaphylococcus aureus alpha hemolysin polypeptide.
 11. The polypeptideof claim 10, wherein the non-native amino acids are tyrosine (Y). 12.The polypeptide of claim 10, wherein the non-native amino acidsubstitution at position 109 is tyrosine (Y) and the non-native aminoacid substitution at position 149 is tryptophan (W).
 13. The polypeptideof claim 9, wherein at least one of the non-native amino acids istryptophan (W).
 14. The polypeptide of claim 9, wherein at least one ofthe non-native amino acids is tyrosine (Y).
 15. The polypeptide of claim9, comprising one or more substitutions at positions that are part ofthe sequence of the beta barrel region, wherein the beta barrel regioncomprises positions 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145 and 147 of a wild-typeStaphylococcus aureus alpha hemolysin polypeptidem and the amino acidresidues of the wild-type Staphylococcus aureus alpha hemolysinpolypeptide align with positions of the amino acid sequence of theStaphylococcus aureus alpha hemolysin polypeptide of SEQ ID NO.
 1. 16.The polypeptide of claim 15, wherein the one or more substitutions atpositions that are part of the sequence of the beta barrel regioncomprises position 135.